CN116964207A

CN116964207A - Methods for enhancing nonviral gene therapy

Info

Publication number: CN116964207A
Application number: CN202180094347.5A
Authority: CN
Inventors: A·那哈维; X·安格拉; R·臧
Original assignee: Spark Treatment Co
Current assignee: Spark Treatment Co
Priority date: 2020-12-23
Filing date: 2021-12-22
Publication date: 2023-10-27
Also published as: JP2024501284A; MX2023007609A; AU2021410086A1; CA3206000A1; EP4267253A1; WO2022140788A1; US20240074986A1

Abstract

The present application describes methods of delivering a transgene to a subject in need thereof. In particular, the method comprises administering to the subject (i) a phagocytic depleting agent, and (ii) a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier. The methods can be used to treat a subject in need of treatment for a disease caused by loss of function or activity of a protein, or to treat a subject in need of treatment for a disease caused by gain of function or expression of a protein.

Description

Methods for enhancing nonviral gene therapy

RELATED APPLICATIONS

The present application claims priority from U.S. provisional patent application No. 63/130,105, filed 12/23 in 2020, which is incorporated herein by reference in its entirety, including all text, tables, and figures.

Technical Field

The present application relates to the field of gene therapy. In particular, it relates to methods of enhancing non-viral gene therapy.

Background

Gene therapy involves the delivery of exogenous nucleic acids to cells to provide therapeutic benefits. Genetic diseases known to be abnormal in pathogenesis can be treated using gene therapy by blocking erroneous or overexpressed genes, or by providing working copies of dysfunctional genes. Such gene therapy methods are currently being developed to treat or cure some diseases caused by genetic abnormalities.

Successful gene therapy requires an effective therapeutic genetic material delivery system. Successful delivery of genetic material requires, for example, that the gene delivery cells be deliverable to a subject, be able to protect the genetic material from enzymatic degradation, have a long life in vivo, be able to reach a desired location in vivo, be biocompatible and biodegradable or have tolerable toxicity, and be able to cross the cell membrane and pass through the cytosol and/or cross the nuclear membrane to release the genetic material at a desired point of action.

Gene therapy can be delivered by viral and non-viral vectors. When recombinant DNA viruses remain episomal or integrated into the chromosome, viral vectors exhibit high transduction efficiency and long-term stable expression of foreign genes. However, their in vivo efficacy may be limited due to concerns about toxicity, random integration of genetic material into the genome, disruption of proper gene function, cancer formation, potential replication, cell division dependent dilution, limited DNA carrying capacity, severe immune and inflammatory responses (in part due to humoral and cell-mediated immune responses to the vector capsid), and failure to re-administer to the subject due to the presence of pre-existing antibodies to the vector capsid in a portion of the subject.

Non-viral gene delivery methods are being developed to overcome the shortcomings of viral vectors. Certain types of non-viral vectors are easier to prepare than viral vectors, do not require replication, and are less likely to generate an adaptive immune response due to the lack of a protein-based vector capsid. In addition, non-viral vectors offer some valuable advantages, including the lack of transgene size restriction associated with viral vectors, the ability to administer to subjects with antibodies pre-existing in the vector capsid, and the ability to re-administer to subjects. However, non-viral delivery methods suffer from challenges such as toxicity, transfection efficiency, nucleic acid degradation, and innate immunity. Furthermore, non-viral methods have demonstrated low efficiency of gene delivery to somatic targets, and in vivo gene expression levels are also lower than viral methods.

The liver is the central organ of metabolism, which is the gene therapy target for many inherited metabolic diseases and disorders, including congenital metabolic defects and hemophilia, and acquired diseases such as liver cancer and hepatitis. The specific cellular target for liver-directed gene therapy is parenchymal hepatocytes (parencgymal liver cell), or hepatoblasts (hepatocytotes). However, nanoparticle uptake by predominantly non-parenchymal cells, including Kupffer cells (50%) and liver sinus endothelial cells (30%), limits the efficacy of liver parenchymal cell-directed gene therapy (Shi et al, J Histochem cytochem.2011 (8): 727-40; jacobs et al, pharmaceuticals 2012,5 (12): 1372-1392). Examples of other tissue-specific macrophages, also known as resident macrophages, include intestinal macrophages in the intestinal tract, microglia in the brain, alveolar macrophages in the lung, resident renal macrophages, skin macrophages, erythromyeleous macrophages in the spleen, and osteoclasts in the bone. By enhancing the entry of non-viral vectors into target cells and reducing the uptake of vectors by non-target cells, increased efficacy of site-specific gene therapy can be achieved.

There remains a need for methods of efficiently, specifically and safely delivering therapeutic genetic material to a target of interest.

Disclosure of Invention

Disclosed herein are methods of delivering a transgene to a subject in need thereof. The methods of the invention can be used to treat a subject in need of treatment for a disease caused by loss of function or activity of a protein, or to treat a subject in need of treatment for a disease caused by gain of function or expression of a protein.

While not wishing to be bound by any theory or particular mechanism, it is believed that depleting phagocytic immune cells by administration of a phagocytic depleting agent may reduce the unwanted uptake of the non-viral vector by the non-target cells, enhance entry of the non-viral vector into the target cells, and result in increased transgene expression.

In certain embodiments, the invention relates to methods of delivering a transgene to a subject in need thereof, the methods comprising:

a. administering a phagocyte depleting agent to a subject; and

b. a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier is administered to a subject.

In certain embodiments, the pharmaceutical composition comprises:

a. Non-viral delivery nanoparticles; and

b. a non-viral vector, wherein the non-viral vector comprises a transgene operably linked to a promoter.

In certain embodiments, the phagocytic depleting agent is a monocyte and/or macrophage depleting agent.

In certain embodiments, the monocyte and/or macrophage depleting agent is a CD115 inhibitor.

In certain embodiments, the CD115 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds CD 115.

In certain embodiments, the anti-CD 115 antibody or antigen-binding fragment thereof is ibritumomab (emituzumab), AMG820, or carbitolizumab (cabiralizumab).

In certain embodiments, the CD115 inhibitor is a small molecule inhibitor of CD 115.

In certain embodiments, the small molecule inhibitor of CD115 is pexidatinib.

In certain embodiments, the monocyte and/or macrophage depleting agent is not a chlorophosphate (clodronate), or the monocyte and/or macrophage depleting agent comprises a chlorophosphate and one or more additional phagocyte depleting agents such as a CD115 inhibitor or a CD177, CD14, CD15, CD11b, CD16, CD32, CD33, CD44, CD45, CD66b, CD18 or CD62L inhibitor.

In certain embodiments, the phagocytic depleting agent is a neutrophil depleting agent.

In certain embodiments, the neutrophil depleting agent is CD177, CD14, CD15, CD11b, CD16, CD32, CD33, CD44, CD45, CD66b, CD18 or CD62L inhibitor, or an agent that inhibits the corresponding or equivalent human protein and/or functional cell type.

In certain embodiments, the CD177, CD14, CD15, CD11b, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L inhibitor is an antibody or antigen binding fragment thereof that specifically binds CD177, CD14, CD15, CD11b, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD 62L.

In certain embodiments, the CD177, CD14, CD15, CD11b, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L inhibitor is a small molecule inhibitor of CD177, CD14, CD15, CD11b, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD 62L.

In certain embodiments, the phagocytic depleting agent is a dendritic cell depleting agent.

In certain embodiments, the non-viral vector and the phagocytic depleting agent are co-administered.

In certain embodiments, the phagocytic depleting agent is administered at least one day prior to administration of the non-viral vector, optionally no more than 1 year prior to administration of the non-viral vector.

In certain embodiments, the method further comprises administering a bisphosphonate to the subject, preferably with one or more other phagocytic depleting agents.

In certain embodiments, the bisphosphonate is a clodronate, pamidronate, ibandronate, or zoledronate, optionally a clodronate.

In certain embodiments, the method further comprises administering an immunosuppressant to the subject.

In certain embodiments, the immunosuppressant is a steroid.

In certain embodiments, the steroid is a corticosteroid, optionally dexamethasone.

In certain embodiments, the method results in expression of the transgene in the subject.

In certain embodiments, the undesired immune response induced by the non-viral vector is minimal or absent in the subject.

In certain embodiments, the non-viral delivery particle is selected from the group consisting of a lipid nanoparticle, a polymer nanoparticle, a protein-based nanoparticle, and a peptide cage.

In certain embodiments, the non-viral vector is a dsDNA molecule, wherein the dsDNA molecule is selected from the group consisting of a micro-loop, a plasmid, an open linear duplex DNA, and a closed linear duplex DNA (CELiD/ceDNA/douggybone DNA).

In certain embodiments, the non-viral vector is a ssDNA molecule, wherein the ssDNA molecule is a closed circular DNA or an open linear DNA.

In certain embodiments, the transgene encodes a therapeutic or prophylactic protein or peptide.

In certain embodiments, the transgene encodes a therapeutic or prophylactic nucleic acid.

Also disclosed herein are combinations or compositions, such as, but not limited to, kits (packages) and kits, having components that can be used to practice the methods of the invention. In certain embodiments, the kit of parts or kits comprises: (a) A pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier; (b) a phagocyte depleting agent; and (c) a label having instructions for performing the method as disclosed herein. In certain embodiments, (a) and (b) are in different or the same containers. In certain embodiments, the combination, kit of parts or kit may further comprise an immunosuppressant.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings. In the drawings:

FIG. 1 shows the results of the immune response of the study described in example 1, in particular FIG. 1A is a graph showing IFN-gamma cytokine production levels in animal plasma 6 hours after administration with 50 μg of DNA-LNP, with or without anti-CD 115 or anti-Ly 6G antibodies or large ODN or H-151; and FIG. 1B is a graph showing IFN-gamma cytokine production levels in animal plasma 6 hours after administration of 10 μg of DNA-LNP with or without anti-CD 115 or anti-Ly 6G antibodies or large ODN or H-151.

FIG. 2 shows the efficacy results of the study described in example 1, in particular FIG. 2A is a graph showing the levels of FIX transgene expression in animal plasma 1, 8, 14, 28, 63, 84 and 98 days after administration with 50 μg of DNA-LNP, with or without anti-CD 115 or anti-Ly 6G antibodies or large ODN or H-151; and FIG. 2B is a graph showing levels of FIX transgene expression in animal plasma 1, 8, 14, 28, 63, 84 and 98 days after administration of 10 μg of DNA-LNP with or without anti-CD 115 or anti-Ly 6G antibody or large ODN or H-151.

FIG. 3 shows the results of the immune response of the study described in example 2, in particular FIG. 3A is a graph showing IFN-gamma cytokine production levels in animal plasma 6 hours after administration with 50 μg of DNA-LNP, with or without anti-CD 115 and/or clodronate; and FIG. 3B is a graph showing IL-6 cytokine production levels in animal plasma 6 hours after administration of 50 μg of DNA-LNP, with or without anti-CD 115 and/or clodronate.

FIG. 4 shows the efficacy results of the study described in example 2, in particular FIG. 4 is a graph showing the levels of FIX transgene expression in plasma of animals 1 week after administration of 50 μg of DNA-LNP, with or without anti-CD 115 and/or clodronate.

Figure 5 shows the depletion efficacy resulting from treatment with clodronate, anti-CD 115 and pexidanib. Fig. 5A provides results of measuring clec4f+ cells. Fig. 5B provides the results of measuring cd68+ cells.

Figure 6 shows the depletion efficacy resulting from treatment with pexidanib mixed feed. Fig. 6A provides the results of measuring cd68+ cells. Fig. 6B provides results of measuring clec4f+ cells.

FIG. 7 shows hFIX expression in mice treated with pexidanib and administered with DNA-LNP-5. Mice were pretreated with pexidanib for 7 or 21 days. The levels of hFIX in mouse plasma were quantified using ELISA 1, 2, 3 or 4 weeks after dosing.

FIG. 8 shows hFIX expression in mice treated with anti-CD-115 antibodies and administered with DNA-LNP-5. Mice were pretreated three times with anti-CD-115 antibody prior to DNA-LNP-5 administration (days-5, -3 and-1). The levels of hFIX in mouse plasma were quantified using ELISA 1, 2, 4 or 12 weeks after dosing. In fig. 8, "ns" indicates that P <0.05 by t-test is insignificant; LLOQ represents the lower limit of quantification.

FIG. 9 shows hFIX expression in mice treated with anti-CD-115 antibodies and administered with DNA-LNP-3. Mice were pretreated four times with anti-CD-115 antibodies (days-10, -8, -6 and-2) prior to DNA-LNP-3 administration. The levels of hFIX in mouse plasma were quantified using ELISA 1, 2, 4 or 12 weeks after dosing. In fig. 9, "ns" indicates that P <0.05, and P <0.01 by t-test are insignificant; LLOQ represents the lower limit of quantification.

FIG. 10 shows hFIX expression in mice treated with anti-CD-115 antibodies and administered with DNA-LNP-4. Mice were pretreated four times with anti-CD-115 antibodies (days-10, -8, -6 and-2) prior to DNA-LNP-4 dosing. The levels of hFIX in mouse plasma were quantified using ELISA 1, 2, 4 or 12 weeks after dosing. In fig. 10, "ns" indicates that P <0.05, and P <0.001 by t-test are insignificant; LLOQ represents the lower limit of quantification.

Detailed Description

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is incorporated by reference herein in its entirety. The discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these materials form part of the prior art with respect to any invention disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms cited herein have the meaning as shown in the specification. All patents, published patent applications, and publications cited herein are hereby incorporated by reference as if fully set forth herein.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term "comprising" as used herein may be replaced with the term "containing" or "including" or sometimes with the term "having" as used herein.

As used herein, "consisting of …" excludes any element, step or ingredient not specified in the claim elements. As used herein, "consisting essentially of …" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims. Any of the above-described terms "comprising," "including," "comprising," and "having" whenever used herein in the context of aspects or embodiments of the present invention may be substituted with the term "consisting of …" or "consisting essentially of …" to change the scope of the present disclosure.

As used herein, the connection term "and/or" between a plurality of referenced elements is understood to encompass both individual and combined options. For example, when two elements are connected by an "and/or", the first option refers to the applicability of the first element without the second element. The second option refers to applicability of a second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and/or" as used herein. Concurrent applicability of more than one option is also understood to fall within the meaning, thus meeting the requirements of the term "and/or".

All features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are examples of one genus of equivalent or similar features.

The term "about" as used herein refers to a value within 10% of the base parameter (i.e., plus or minus 10%). For example, "about 1:10" means 1.1:10.1 or 0.9:9.9, and about 5 hours means 4.5 hours or 5.5 hours, etc. The term "about" at the beginning of a string of values modifies each value by 10%.

All numbers or numerical ranges include integers within those ranges as well as values or fractions of integers within the range unless the context clearly indicates otherwise. Thus, for purposes of illustration, reference to a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, etc., as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. Thus, also for purposes of illustration, reference to numerical ranges such as "1-4" includes 2, 3, and 1.1, 1.2, 1.3, 1.4, etc. For example, "1 to 4 weeks" includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

Further, reference to numerical ranges such as "0.01 to 10" includes 0.011, 0.012, 0.013, and the like, and 9.5, 9.6, 9.7, 9.8, 9.9, and the like. For example, a dose of about "0.01mg/kg to about 10mg/kg" of subject body weight includes 0.011mg/kg, 0.012mg/kg, 0.013mg/kg, 0.014mg/kg, 0.015mg/kg, etc., and 9.5mg/kg, 9.6mg/kg, 9.7mg/kg, 9.8mg/kg, 9.9mg/kg, etc.

References to more (greater than) or less than an integer include any number greater or less than the referenced number, respectively. Thus, for example, reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and the like. For example, the "2 or more times" of administration of a non-viral vector and/or a phagocytic depleting agent includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times.

Further, reference to numerical ranges such as "1 to 90" includes 1.1, 1.2, 1.3, 1.4, 1.5, and the like, and 81, 82, 83, 84, 85, and the like. For example, "between about 1 minute and about 90 days" includes 1.1 minute, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as 1 day, 2 days, 3 days, 4 days, 5 days … days, 82 days, 83 days, 84 days, 85 days, etc.

To assist the reader of this application, this specification has been divided into different paragraphs or sections or directed to certain embodiments of the application. These separations should not be construed as separating the essence of one paragraph or part or embodiment from the essence of another paragraph or part or embodiment. In contrast, those skilled in the art will understand that the present description has broad application and encompasses all combinations of parts, paragraphs and sentences that may be considered. The discussion of any embodiment is merely exemplary and is not intended to represent that the scope of the disclosure, including the claims, is limited to these examples.

Provided herein are methods of improving gene therapy comprising administering an agent that modulates immune cells. The methods of the application can be used to treat a subject in need of treatment for a disease, such as a disease caused by loss of function or activity of a protein, or a disease caused by gain of function or expression of a protein.

In a general aspect, provided herein is a method of delivering a transgene to a subject in need thereof, comprising:

a. administering a phagocyte depleting agent to a subject; and

In certain embodiments, the pharmaceutical composition comprises:

a. non-viral delivery nanoparticles; and

Gene transfer system

The term "vector" or "gene transfer vector" as used herein refers to a nucleic acid molecule comprising a gene of interest. Examples of vectors include, but are not limited to, viral vectors delivered by viral particles or virus-like particles (VLPs) that are similar to viral particles but are not infectious, such as retroviruses, adenoviruses, adeno-associated viruses, and lentiviral particles or VLPs; and non-viral vectors delivered by non-viral gene transfer systems such as microinjection, electroporation, liposomes, large natural polymers, large synthetic polymers, and polymers comprising both natural and synthetic components.

As used herein, "non-viral vector" refers to a vector that is not delivered by a viral particle or virus-like particle (VLP). According to certain embodiments, the non-viral vector is a vector that is not delivered through the capsid. The carrier may be encapsulated, mixed or otherwise associated with the non-viral delivery nanoparticle.

As used herein, the term "gene transfer system" refers to any means of delivering a composition comprising a nucleic acid sequence (e.g., a transgene) to a cell or tissue. For example, the gene transfer system may be a viral gene transfer system, e.g., intact viruses, modified viruses, and VLPs, to facilitate delivery of the viral vector to a desired cell or tissue. The gene transfer system may also be a non-viral delivery system that does not comprise viral coat proteins or form viral particles or VLPs, e.g., liposome-based systems, polymer-based systems, protein-based systems, metal particle-based systems, peptide cage systems, and the like.

Any suitable non-viral delivery system known to those of skill in the art in view of this disclosure may be used with the present invention. The non-viral delivery nanoparticle may be, for example, a lipid-based nanoparticle, a polymer-based nanoparticle, a protein-based nanoparticle, a microparticle, a microcapsule, a metal particle-based nanoparticle, a peptide cage nanoparticle, or the like.

The non-viral delivery nanoparticles of the invention may be constructed by any method known in the art, and the non-viral vectors of the invention comprising the non-viral delivery nanoparticles and the nucleic acid molecules comprising the therapeutic transgene may be constructed by any method known in the art.

Lipid-based delivery systems

Lipid-based delivery systems are well known in the art, and any suitable lipid-based delivery system known to those skilled in the art in view of the present disclosure may be used in the present invention. Examples of lipid-based delivery systems include, for example, liposomes, lipid nanoparticles, micelles, or extracellular vesicles.

"lipid nanoparticle" or "LNP" refers to lipid-based vesicles useful for delivery of nucleic acid molecules that are nanoscale in size, i.e., from about 10nm to about 1000nm, or from about 50nm to about 500nm, or from about 50nm to about 200nm.

The DNA is negatively charged. Thus, this may be advantageous for LNP to comprise cationic lipids, e.g., amino lipids. Exemplary amino lipids have been described in U.S. patent nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, and 7,745,651, and U.S. patent publication nos. 2016/0213785, 2016/0199485, 2015/0265708, 2014/0288146, 2013/012338, 2013/016307, 2013/0064894, 2012/0172411, and 2010/017125, all of which are incorporated herein by reference in their entirety. In certain embodiments, the LNP comprises an amino lipid, such as any of those described in WO 2013/063268, which is incorporated herein in its entirety.

The terms "cationic lipid" and "amino lipid" are used interchangeably herein to include those lipids and salts thereof having one, two, three or more fatty acids or fatty alkyl chains and a pH titratable amino group (e.g., alkylamino or dialkylamino). Cationic lipids are typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid, and are substantially neutral at a pH above the pKa. The cationic lipid may also be a titratable cationic lipid. In certain embodiments, the cationic lipid comprises: a protonatable tertiary amine (e.g., pH titratable) group; a C18 alkyl chain, wherein each alkyl chain independently has 0-3 (e.g., 0, 1,2, or 3) double bonds; and ether, ester or ketal linkages between head groups (head groups) and alkyl chains.

The cationic lipids may include, but are not limited to, 1,2-dioleyloxy-N, N-dimethylaminopropane (1, 2-dioleyleveloplane, DLinDMA), 1,2-dioleyloxy-N, N-dimethylaminopropane (1, 2-dioleylenoyl-N, N-dimethylaminopropane, DLenDMA), 1, 2-di-gamma-linoleylenoyloxy-N, N-dimethylaminopropane (1, 2-di-y-linolenyloxy-N, N-dimethylaminopropane,. Gamma. -DLenDMA), 2-dioleyledo-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (2, 2-dimethyiminoethyl) - [1,3] -dioxolane, DLin-K-C2-DMA, also known as DLin-C2K-DMA, XTC2 and C2K), 2-diimine-4-dimethylaminomethyl- [1,3] -dioxolane (2, 2-diimine-4-dimethylol-1, 3] -dioxane, DLin-K-DMA), diimine-3-dimethylaminomethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC 2), (6Z, 9Z,28Z, 31Z) -triacontan-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butyrate ((6Z, 9Z, 28Z) -hexata-6,9,28,31-tetraen-19-yl-4- (dimethylamine) butyrate, DLin-M-C3-DMA, also known as MC 3), salts thereof and mixtures thereof. Other cationic lipids also include, but are not limited to, 1,2-distearyloxy-N, N-dimethyl-3-aminopropane (1, 2-distearyloxy-N, N-dimethyl-3-aminopropane, DSDMA), 1,2-dioleyloxy-N, N-dimethyl-3-aminopropane (1, 2-dioleyloxy-N, N-dimethyl-3-aminopropane, DODMA), 2-diiodol-4- (3-dimethylaminopropyl) - [1,3] -dioxolane (2, 2-dioleyl-4- (3-dimethylaminoopyl) - [1,3] -dioxolane, DLin-K-C3-DMA), 2-diiodol-4- (3-dimethylaminobutyl) - [1,3] -dioxolane (2, 2-dioleyl-4- (3-dimethylaminobutyl) - [1,3] -dioxolane, DLin-K-C4-DMA), DLen-C2K-DMA, γ -DLen-C2K-DMA, and (DLin-MP) (also referred to as 1-B11).

Further cationic lipids may include, but are not limited to, 2-diiodol-5-dimethylaminomethyl- [1,3] -dioxane (2, 2-diiodol-5-dimethyllaminomethyl- [1,3] -dioxane, DLin-K6-DMA), 2-diiodol-4-N-methylpiperazine- [1,3] -dioxolane (2, 2-diiodol-4-N-methylpiperazine- [1,3] -dioxane, DLin-K-MPZ), 1, 2-diiodol carbamoyloxy-3-dimethylaminopropane (1, 2-diiodol-3-dimethylaminopropane), 1, 2-diiodol-3- (dimethylamino) acetoxypropane (1, 2-diiodol-3- (dimethylamino) propane, DLin-DAC), 1, 2-diiodoxy-3-morpholinopropane (1, 2-diiodoxy-3-morpholinopropane, DLin-MA), 1, 2-diiodoxy-3-dimethylaminopropane (1, 2-diiodoyl-3-dimethylaminopropane, DLinDAP), 1, 2-diiodoxy-3-dimethylaminopropane (1, 2-diiodoxy-3-dimethylaminopropane, DLin-S-DMA), 1-linoleoyl-2-linoleoxy-3-dimethylaminopropane (1-linoleoyl-2-linolexy-3-dimethylaminopropane, DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride (1, 2-diiodoleyloxy-3-trimethylaminopropane chloride salt, DLin-TMA. Cl), 1, 2-diileoyl-3-trimethylaminopropane chloride (1, 2-diileoyl-3-trimethylaminopropane chloride salt, DLin-TAP. Cl), 1, 2-diileyloxy-3- (N-methylpiperazine) propane (1, 2-diileyloxy-3- (N-methylpiperazine) propane, DLin-MPZ), 3- (N, N-diileylamino) -1,2-propanediol (3- (N, N-diileylamino) -1,2-propanediol, DLinaP), 3- (N, N-diileylamino) -1,2-propanediol (3- (N, N-diileylamino) -1,2-propanediol, DOAP), 1, 2-diileyloxy-3- (N, N-diileyloxy) -3- (N-diileylamino) -1,2-propanediol, 3- (N, N-diileylamino) -1,2-propanediol (3- (N, N-diileylamino) -1, 2-propanediol), 1, 2-diileyloxy-3- (N, N-diileylamino) -2-propanediol (3- (N, N-diileylamino) -1,2-propanediol (2, N, N-diileyloxy-2-propanediol) methyl-3- (N, N, N-diileyloxy-2-propanediol) methyl-3- (N, N-diileyloxy-2-trimethyl-3-trimethyl-propane (1, N-2-diileyl-2-trimethyl-carbonyl chloride), n, N-trimethylammonium chloride, DOTMA), N, N-distearoyl-N, N-dimethylammonium bromide (N, N-distearyl-N, N-dimethylammonium bromide, DDAB), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (N- (1- (2, 3-dioleyloxy) propyl) -N, N-trimethylammonium chloride, DOTAP), 3- (N- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (3- (N- (N ', N' -dimethylminethane) -carbamoyl) Chol-e, DC-Chol), N- (1, 2-dimyristoyloxy propan-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (N- (1, 2-dimyristoxyprop-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide, DMRIE), 2,3-dioleyloxy-N- [2 (spermine carboxamide) ethyl ] -N, N-dimethyl-1-propylamine trifluoroacetate (2, 3-dioleylaxy-N- [2 (spermine-carboxamide) ethyl ] -N, N-dimethyl-1-propanamine fluoride acetate, DOSPA), octacosaminoglycinamide (dioctadecylamidoglycyl spermine, DOGS), 3-dimethylamino-2- (cholest-5-en-3- β -oxybutan-4-yloxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (3-dimethylamino-2- (cholest-5-en-3-betaoxybutan-4-yloxy) -1- (cis, cis-9,12-octadecadienoxy propane, CLinDMA), 2- [5'- (cholest-5-en-3- β -oxy) -3' -oxapentaoxo) -3-dimethyl-1- (cis, cis-9',1-2' -octadecadienyloxy) propane (2- [5'- (cholest-5-en-3-beta-oxy) -3' -oxo) -3-dimethyl-1- (cis, cis-9',1-2' -octacams-3-dimethylaminopropane (CpLinDMA), N-dimethyl-3, 4-Dioleoxybenzylamine (DMOBA), 1,2-N, N '-dioleylcarbamoyl-3-dimethylaminopropane (1, 2-N, N' -dioleylarbamamyl-3-dimethylaminopropane), 1,2-N, N '-dioleylcarbamoyl-3-dimethylaminopropane (1, 2-N, N' -dioleylcarbamyl-3-dimethylaminopropane, DLincarbDAP), dexamethasone-spermine (DS), and disubstituted spermine (D2S), or mixtures thereof.

Many cations can be usedCommercial preparations of ionic lipids, e.g.(including DOTMA and DOPE, available from GIBCO/BRL) and +.>(comprising DOSPA and DOPE available from GIBCO/BRL).

Other commercially available ionizable lipids that may be used include, for example, SS-OP (NOF U.S. Co.), C12-200 (described in Kauffman et al, nano Lett.2015,15,11,7300-7306, incorporated herein by reference), 306Oi10, CKK-E12, MC3, branched-CKK-E12, lipid 5, lipid 9, ATX-002, and ATX-003 (described in Payne and Chivukula, international publication No. WO2015074085, incorporated herein), SM-102 (Cayman Chemicals); ALC-0315 (Selleck Chemicals) and Merck-32.

In certain embodiments, the amount of cationic lipid can be from about 10% molar ratio of LNP to about 85% molar ratio of lipid nanoparticle, or from about 50% molar ratio of LNP to about 75% molar ratio of LNP.

Sterols may impart fluidity to LNP. As used herein, "sterols" refers to any naturally occurring sterols of plant (plant sterols) or animal (animal sterols) origin as well as non-naturally occurring synthetic sterols, all of which are characterized by the presence of a hydroxyl group at the 3-position of the steroid a-ring. The sterol may be any sterol conventionally used in the art of liposome, lipid vesicle or lipid particle preparation, most commonly cholesterol. The phytosterols may include campesterol, sitosterol and stigmasterol. Sterols also include sterol-modified lipids such as those described in U.S. patent application publication 2011/0177156. In certain embodiments, the amount of sterols may be from about 5 wt% of the LNP to about 50 wt% of the lipid nanoparticle or from about 10 wt% of the LNP to about 25 wt% of the LNP.

LNP may comprise neutral lipids. Neutral lipids can comprise any lipid species that exists in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, but are not limited to, diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalins, and cerebrosides. The selection of neutral lipids is generally guided by, inter alia, consideration of particle size and necessary stability. In certain embodiments, the neutral lipid component may be a lipid having two acyl groups (e.g., diacyl phosphatidylcholine and diacyl phosphatidylethanolamine).

Lipids having acyl chain groups of various chain lengths and saturations are available or can be isolated or synthesized by well known techniques. In certain embodiments, lipids containing saturated fatty acids having carbon chain lengths in the range of C14-C22 may be used. In certain embodiments, lipids having monounsaturated fatty acids or di-unsaturated fatty acids with carbon chain lengths in the range of C14-C22 are used. In addition, a lipid having a mixture of saturated fatty acid chains and unsaturated fatty acid chains may be used. Exemplary neutral lipids include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), or any related phosphatidylcholine. Neutral lipids may also consist of sphingomyelin, dihydrosphingomyelin or phospholipids with other head groups such as serine and inositol.

In certain embodiments, the amount of neutral lipid can be from about 0.1 wt% of the lipid nanoparticle to about 99 wt% of the LNP, or from about 5 wt% of the LNP to about 15 wt% of the LNP, such as about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

The LNP-encapsulated nucleic acid molecules can be incorporated into pharmaceutical compositions, e.g., pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions are useful, among other things, for in vivo or ex vivo administration and delivery of LNP-encapsulated nucleic acid molecules to a subject.

The preparation of LNP may be combined with additional components, which may include, for example, but are not limited to, polyethylene glycol (PEG) and sterols.

The term "PEG" refers to a linear, water-soluble polymer of ethylene PEG repeat units having two terminal hydroxyl groups. PEG is classified by its molecular weight; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, while PEG 5000 has an average molecular weight of about 5,000 daltons. PEG is commercially available from Sigma Chemical co. And other companies, including, for example, the following functional PEG: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH 2), monomethoxy polyethylene glycol-trisulphonate (monomethoxypolyethylene glycol-tresylate, mePEG-TRES) and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

In certain embodiments, the PEG may be polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons, and optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments, PEG may be substituted with methyl at the terminal hydroxyl position. In certain embodiments, the average molecular weight of the PEG may be from about 750 to about 5,000 daltons, or from about 1,000 to about 5,000 daltons, or from about 1,500 to about 3,000 daltons, or from about 2,000 daltons or about 750 daltons. The PEG may be optionally substituted with alkyl, alkoxy, acyl, or aryl. In certain embodiments, the terminal hydroxyl groups may be substituted with methoxy or methyl.

PEG modified lipids include the PEG-dialkyloxypropyl conjugates (PEG-DAA) described in U.S. Pat. Nos. 8,936,942 and 7,803,397. Useful PEG-modified lipids (or lipid-polyoxyethylene conjugates) may have various "anchoring" lipid moieties to immobilize the PEG moiety on the surface of the lipid vesicle. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates described in U.S. patent No. 5,820,873 (e.g., PEG-CerC14 or PEG-CerC 20), PEG-modified dialkylamines and PEG-modified 1, 2-diacyloxypropane-3-amines. In certain embodiments, the PEG-modified lipids can be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, the amount of PEG may be from about 0.5 wt% of the LNP to about 20 wt% of the LNP, or from about 5 wt% of the LNP to about 15 wt% of the LNP.

Furthermore, the LNP may be PEG-modified and/or sterol-modified LNP. The LNP may be the same or different LNP in combination with additional components. In other words, the same LNP may be PEG-modified and sterol-modified, or the first LNP may be PEG-modified and the second LNP may be sterol-modified. Optionally, the first modified LNP and the second modified LNP can be combined.

In certain embodiments, the LNP may range in size from about 10nm to 500nm, or from about 50nm to about 200nm, or from 75nm to about 125nm, prior to encapsulation.

In certain embodiments with respect to the LNP, the LNP is described by Billingsley et al, nano Lett.2020,20,1578 or International patent publication WO2021/077066 (both incorporated herein by reference in their entirety). Billingsley et al and WO2021/077066 describe LNPs containing lipid anchored PEG, cholesterol, phospholipids and ionizable lipids. In certain embodiments, the LNP contains a C14-4 polyamine core and/or has a particle size of about 70 nm. C14-4 has the following structure.

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In certain embodiments with respect to LNP, the LNP consists of a cationic lipid or lipopeptide described in U.S. patent No. 10,493,031, U.S. patent No. 10,682,374, or WO2021/077066 (each of which is incorporated herein by reference in its entirety). In certain embodiments, the LNP contains a cationic lipid, a cholesterol-based lipid, and/or one or more PEG-modified lipids. In certain embodiments, the LNP contains cKK-E12 (Dong et al, PNAS2014,111 (11), 3955):

In certain embodiments with respect to the LNP, the LNP comprises a Lipid 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described in Sabnis et al, molecular Therapy, vol.26, no.6,1509-1519 (incorporated herein by reference in its entirety). In certain embodiments, the LNP comprises Lipid 5, 8, 9, 10, or 11 described by Sabnis et al. Lipid 9 has the structure:

other lipids that may be utilized include Roces et al, pharmaceuticals 2020,12,1095; jayaraman et al; angel.chem.int.ed.2012, 51,8529-8533; and Maier et al www.moleculartherapy.org Vol.21, no.8,1570-1578,2013; liu et al, adv. Mate.2019, 31,1902575, e.g., bama-O16B); cheng et al, adv. Mate.2018, 30,1805308, e.g., 5A2-SC8; hajj and Ball, small 2019,15,1805097, e.g., 306Oi10 (each incorporated by reference herein in its entirety); and Du et al, patent application publication number US2016/0376224, e.g., compounds 1-46, as shown in table 1 below).

In certain embodiments, the LNP contains one or more lipids as provided in table 1, or a pharmaceutically acceptable salt thereof.

TABLE 1

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In certain embodiments, the LNP comprises the following components in mol%: about 20% to 65% of one or more cationic lipids, about 1% to about 50% of one or more phospholipid lipids, about 0.1% to 10% of one or more PEG conjugated lipids, and about 0% to about 70% cholesterol; about 20% to 50% of one or more cationic lipids, about 5% to about 20% of one or more phospholipid lipids, about 0.1% to 5% of one or more PEG conjugated lipids, and about 20% to about 60% cholesterol; in further embodiments, the phospholipid lipid is a neutral lipid; and the phospholipid lipid is DOPE or DSPC.

Polymer-based systems

Polymer-based delivery systems are well known in the art, and any suitable polymer-based delivery system or polymer nanoparticle known to those skilled in the art in view of this disclosure may be used in the present invention. The DNA may be embedded in the polymer matrix of the polymer nanoparticle or may be adsorbed or conjugated to the surface of the nanoparticle. Examples of polymers commonly used for gene delivery include, for example, polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), poly (ethyleneimine) (PEI), chitosan, dendrimers, polyanhydrides, polycaprolactone, and polymethacrylates.

The polymer-based non-viral vectors can have different sizes ranging from about 1nm to about 1000nm, optionally from about 10nm to about 500nm, optionally from about 50nm to about 200nm, optionally from about 100nm to about 150nm, optionally about 150nm or less.

Protein-based systems

Protein-based delivery systems are well known in the art, and any suitable protein-based delivery system or Cell Penetrating Peptide (CPP) known to those of skill in the art in view of this disclosure may be used in the present invention.

CPPs are short peptides (6-30 amino acid residues) with potential intracellular penetration ability to deliver therapeutic molecules. Most CPPs consist primarily of arginine and lysine residues, rendering them cationic and hydrophilic, but CPPs may also be amphiphilic, anionic or hydrophobic. CPP can be derived from a natural biomolecule (e.g., tat, an HIV-1 protein) or obtained synthetically (e.g., poly-L-lysine, poly-arginine) (Singh et al., drug Deliv.2018;25 (1): 1996-2006). Examples of CPPs include, for example, cationic CPPs (high positive charges) (e.g., tat peptide, pentatin, protamine, poly-L-lysine, polyarginine, etc.); amphiphilic CPP (chimeric or fusion peptide, constructed from different sources, containing positively and negatively charged amino acid sequences) (e.g., transportan, VT)5. Antibacterial peptide (bactenecin) -7 (Bac 7), proline-rich peptide (PPR), SAP (VRLPPP) ₃ TP10, pep-1, MPG, etc.); membranotopic (CPP) that exhibits both hydrophobic and amphiphilic properties and contains large aromatic residues and small residues (e.g., gH625, SPION-PEG-CPP NP, etc.); and hydrophobic CPPs (containing only non-polar motifs or residues) (e.g., SG3, pfvyl i, pep-7, fibroblast Growth Factor (FGF), etc.).

The protein-based non-viral vectors may have different sizes ranging from about 1nm to about 1000nm, optionally from about 10nm to about 500nm, optionally from about 50nm to about 200nm, optionally from about 100nm to about 150nm, optionally about 150nm or less.

Peptide cage system

Peptide cage based delivery systems are well known in the art and any suitable peptide cage based delivery system known to those skilled in the art in view of this disclosure may be used in the present invention. In general, any protein material capable of being assembled into a cage-like structure to form a constrained internal environment may be used. Several different types of protein "shells" can be assembled and loaded with different types of materials. For example, protein cages comprising a viral coat protein shell encapsulating a non-viral material (e.g., from cowpea chlorotic mottle virus (Cowpea Chlorotic Mottle Virus, CCMV) protein shells), as well as protein cages formed from non-viral proteins, have been described (see, e.g., U.S. patent nos. 6,180,389 and 6,984,386, U.S. patent application 20040028694, and U.S. patent application 20090035389, incorporated herein in their entirety). The peptide cage may comprise a protein shell that self-assembles to form a protein cage (e.g., a structure with internal cavities that may naturally enter the solvent or may be brought into the solvent by changing the solvent concentration, pH, equilibrium ratio).

Examples of protein cages derived from non-viral proteins include, for example, ferritin and deferiprone, derived from eukaryotic and prokaryotic species, for example, 12 and 24 subunit ferritin; and protein cages formed from Heat Shock Proteins (HSP), for example, 24 subunit heat shock proteins forming an internal core space, small HSP of Methanococcus jannaschii (Methanococcus jannaschii), dodecamer (dodecameric) Dsp HSP of E.coli, mrgA protein, and the like. As understood by those skilled in the art, monomers of the protein cage may be naturally occurring, or may be in variant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments) that may be made.

The protein cages may have different core sizes ranging from about 1nm to about 1000nm, optionally from about 10nm to about 500nm, optionally from about 50nm to about 200nm, optionally from about 100nm to about 150nm, optionally about 150nm or less.

Therapeutic nucleic acid

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, antisense DNA/RNA, plasmid DNA, linear DNA, (polynucleotides and oligonucleotides), chromosomal DNA, spliced or non-spliced mRNA, rRNA, tRNA inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-spliced RNA or antisense RNA), locked nucleic acid analogs (LNA), oligonucleotide DNA (ODN) single and double stranded, immunostimulatory sequences (ISS), riboswitches, and ribozymes.

Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. The nucleic acid may be single-stranded, double-stranded or triple-stranded, linear or circular, and may be of any length. In discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing sequences in the 5 'to 3' direction.

In certain embodiments, the nucleic acid agent is a single-stranded (ssDNA) or double-stranded DNA (dsDNA) molecule. In certain embodiments, the nucleic acid agent is used for therapeutic purposes, e.g., ssDNA or dsDNA encoding a therapeutic transgene. In certain embodiments, the dsDNA molecule is a micro-loop, a nano-plasmid, an open linear duplex DNA, or a closed-end linear duplex DNA (CELiD/ceDNA/doughybone DNA). In certain embodiments, the ssDNA molecule is closed circular DNA or open linear DNA.

According to the present invention, ssDNA and dsDNA molecules comprising therapeutic transgenes may be prepared by conventional techniques well known to those skilled in the art.

"transgene" is used herein to conveniently refer to a nucleic acid that is intended to or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a heterologous polynucleotide sequence or heterologous nucleic acid encoding a protein or peptide or nucleic acid (e.g., miRNA, etc.). The terms transgene and heterologous nucleic acid/polynucleotide sequence are used interchangeably herein.

Typically, a "therapeutic transgene" of the invention comprises an expression cassette. As used herein, the term "expression cassette" refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a nucleic acid molecule of the invention. Typically, the expression cassette comprises a nucleic acid molecule of the invention operably linked to a promoter sequence. The term "operably linked" refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of the coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to the regulatory sequence in a sense or antisense orientation. In certain embodiments, the promoter is a heterologous promoter. As used herein, the term "heterologous promoter" refers to a promoter that is not found in nature operably linked to a given coding sequence.

As used herein, the term "operably linked" means that a regulatory sequence that affects the expression of a transgene is placed at a position relative to the sequence so as to affect the expression of the transgene. This same definition is sometimes also applied to the arrangement of transgene and transcription control elements (e.g., promoters, enhancers, promoters/enhancers and termination elements) in an expression vector. The transgene may be operably linked to regulatory sequences in sense or antisense orientation. Various regulatory sequences that can be operably linked to a transgene are known in the art and can be used in the methods of the invention. In certain embodiments, the operably linked regulatory sequence is a promoter, such as a liver-specific promoter, or a promoter/enhancer, such as ApoE/hAAT.

In certain embodiments, the expression cassette may comprise additional elements, e.g., introns, enhancers, polyadenylation sites, woodchuck response elements (woodchuck response element, WRE), and/or other elements known to affect the level of the coding sequence. As used herein, the term "promoter" refers to a nucleotide sequence capable of controlling expression of a coding sequence or functional RNA. In general, the nucleic acid molecules of the invention are located 3' to the promoter sequence. In certain embodiments, the promoter sequence consists of proximal and more distal upstream elements, and may comprise enhancer elements. An "enhancer" is a nucleotide sequence that can stimulate promoter activity. Enhancer elements are typically located upstream of the promoter element, but may also be located downstream or within the promoter, e.g., may be an inherent element of the promoter or an inserted heterologous element, to enhance the level or tissue specificity of the promoter. In certain embodiments, the expression cassette comprises a tissue-specific enhancer. In certain embodiments, the promoter is entirely derived from a native gene. In certain embodiments, the promoter consists of different elements derived from different naturally occurring promoters. In certain embodiments, the promoter comprises a synthetic nucleotide sequence. Those skilled in the art will appreciate that different promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions, or in the presence or absence of a drug or transcription cofactor. Ubiquitous, cell type-specific, tissue-specific, developmental stage-specific, and conditional promoters, for example, drug-responsive promoters (e.g., tetracycline-responsive promoters) are well known to those skilled in the art. Examples of promoters include, but are not limited to, phosphoglycerate Kinase (PKG) promoter, CAG (CMV enhancer, a complex of chicken beta actin promoter (CBA) and rabbit beta globin intron), NSE (neuron specific enolase), synaptosin or NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes Simplex Virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), SFFV promoter, rous Sarcoma Virus (RSV) promoter, synthetic promoter, hybrid promoter, and the like. Other promoters may be of human origin, or from other species, including from mice. Common promoters include, for example, the human Cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the rous sarcoma virus long terminal repeat, the β -actin promoter, the rat insulin promoter, the phosphoglycerate kinase promoter, the human α -1 antitrypsin (hAAT) promoter, the transthyretin promoter, the thyroxine-binding globulin (TBG) promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EF1- α promoter, the CAG promoter and other constitutive promoters, hybrid promoters with multiple tissue specificities, neuronal-specific promoters such as synaptocins and glyceraldehyde-3-phosphate dehydrogenase promoters, all of which are well known and readily available to those skilled in the art, may be used to obtain high levels of expression of the coding sequences of interest. In addition, sequences derived from non-viral genes such as mouse metallothionein genes may also be used herein. Such promoter sequences are commercially available from, for example, stratagene (san Diego, calif.). Operably linking the transgene to a tissue-specific regulatory element provides at least partial tissue tropism for transgene expression. In certain embodiments, the transgene is operably linked to a liver-specific promoter and/or liver-specific enhancer. Reference to "liver-specific" means at least part of the tissue tropism of transgene expression in the liver. References to "promoter/enhancer" refer to a promoter and an enhancer operably linked. Examples of promoters active in the liver include, but are not limited to, transthyretin (TTR) gene promoters and mutant forms thereof (Anguela and Shen, U.S. Pat. No. 11,168,124, anguela and Shen, international publication No. WO2017/075619;Costa et al, 1991,Nucleic Acids Research,Vol.19,No.154139-4145); human alpha 1-antitrypsin (hAAT) promoter (Hafenrich et al, blood 1994, vol 84, no. 10.3394-3404); an apolipoprotein a-I promoter; albumin (Miyatake et al, J.Virol.,1997, 71:5124-32); hepatitis B virus core promoter (Sandig et al, gene Ther.1996, 3:1002-9), alpha Fetoprotein (AFP) (Arbuthnot et al, hum. Gene. Ther.,1996, 7:1503-14); a human factor IX promoter; thyroxine-binding globulin (TBG) promoter. Examples of enhancers active in the liver are apolipoprotein E (apoE) HCR-l and HCR-2 (Allan et al, J.biol.chem.,1997, 272:2913-19). In certain embodiments, the promoter/enhancer is ApoE/hAAT (Okuyama et al, 1996,Human Gene Therapy 7:637-645, and Miao et al, 2000,Molecular Therapy Vol.1,No.6). (each reference in this paragraph is incorporated herein by reference.)

In certain embodiments, the expression cassette comprises a suitable secretion signal sequence that allows for secretion of the polypeptide encoded by the nucleic acid molecule of the invention. As used herein, the term "secretion signal sequence" or variant thereof refers to an amino acid sequence that has the function of enhancing (as defined above) secretion of an operably linked polypeptide from a cell, as compared to the secretion level of the native polypeptide. "enhanced" secretion means that the relative proportion of polypeptide secreted from the cell that is synthesized by the cell is increased; the absolute amount of secreted protein does not have to be increased as well. In certain embodiments, substantially all (i.e., at least 95%, 97%, 98%, 99% or more) of the polypeptides are secreted. However, it is not necessary that substantially all or even a substantial portion of the polypeptide is secreted, so long as the level of secretion is enhanced as compared to the native polypeptide. Generally, the secretion signal sequence is cleaved within the endoplasmic reticulum, and in certain embodiments, the secretion signal sequence is cleaved prior to secretion. However, the secretion signal sequence need not be cleaved, so long as secretion of the polypeptide from the cell is enhanced, and the polypeptide has functionality. Thus, in certain embodiments, the secretion signal sequence is partially or fully retained. The secretion signal sequence may be derived in whole or in part from the secretion signal of the secreted polypeptide (i.e., from the precursor) and/or may be synthesized in whole or in part. The length of the secretion signal sequence is not critical; generally, the known secretion signal sequences are about 10-15 to 50-60 amino acids in length. In addition, the known secretion signal from a secreted polypeptide may be altered or modified (e.g., by amino acid substitutions, deletions, truncations, or insertions) so long as the resulting secretion signal sequence is capable of enhancing secretion of the operably linked polypeptide. The secretion signal sequence of the present invention may comprise, consist essentially of, or consist of a naturally occurring secretion signal sequence or modification thereof (as described above). Many secreted proteins and sequences are known in the art that are secreted directly from cells. The secretion signal sequences of the invention may also be wholly or partially synthetic or artificial. Synthetic or artificial secretion signal peptides are known in the art, see, e.g., barash et al, biochem. Biophys. Res. Comm.294:835-42 (2002). In certain embodiments, the invention includes a method of treating a disease by gene therapy using a therapeutic transgene.

In certain embodiments, the heterologous polynucleotide encodes GAA (acid α -glucosidase) for use in treating Pompe disease (Pompe disease) or another glycogen storage disease; ATP7B (copper transport ATPase 2) for the treatment of Wilson's disease; alpha galactosidase a for use in the treatment of briy's disease; ASS1 (arginine succinate synthase) for the treatment of citrullinemia type 1; beta-glucocerebrosidase for the treatment of Gaucher disease (Gaucher disease) type 1; beta-hexosaminidase a for the treatment of tay-saxophone disease (Tay Sachs disease); SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for the treatment of Hereditary Angioedema (HAE), also known as C1 inhibitor deficiency type I and II); or glucose-6-phosphatase for use in the treatment of glycogen storage disease type I (GSDI).

In certain embodiments, the heterologous polynucleotide encodes insulin, glucagon, growth Hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle Stimulating Hormone (FSH), luteinizing Hormone (LH), human chorionic gonadotrophin (hCG), vascular Endothelial Growth Factor (VEGF), angiopoietin, angiostatin, granulocyte Colony Stimulating Factor (GCSF), erythropoietin (EPO), connective Tissue Growth Factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal Growth Factor (EGF), transforming growth factor alpha (tgfα), platelet-derived growth factor (PDGF), insulin growth factor I or II (IGF-I or IGF-II), tgfβ, activin, inhibin, bone Morphogenic Protein (BMP), nerve growth factor (BDNF), brain-derived neurotrophic factor (BDNF), neurotrophin NT 3 or NT4/5, ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), nerve rank protein (neurin), liver-guide factor (hgin), liver-guide factor (HGF), noggin-1 or noggin (noggin-2), or noggin (noggin), sonic hedgehog or tyrosine hydroxylase.

In certain embodiments, the heterologous polynucleotide Thrombopoietin (TPO), interleukins (IL-1 through IL-36, etc.), monocyte chemotactic proteins, leukemia inhibitory factors, granulocyte-macrophage colony stimulating factor, fas ligand, tumor necrosis factor alpha or beta, interferon alpha, beta or gamma, stem cell factor, flk-2/flt3 ligand, igG, igM, igA, igD or IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I or class II MHC molecules.

In certain embodiments, the heterologous polynucleotide encodes CFTR (cystic fibrosis transmembrane regulator), factor (blood coagulation) (factor XIII, factor IX (FIX), factor VIII (FVIII), factor X, factor VII, factor VIIa, protein C, etc.), functionally acquired clotting factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE 65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, beta-globin, alpha-globin, ghost protein, alpha-antitrypsin, adenosine Deaminase (ADA), metal transporter (ATP 7A or ATP 7), sulfonamide enzymes, enzymes involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, beta-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched ketoacid dehydrogenase, hormone, growth factor, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factors-3 and-4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factors alpha and beta, cytokine, alpha-interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug-resistant protein, tumor suppressor proteins (e.g., p53, rb, wt-1, NF1, hippel-Lindau protein (VHL), colon adenomatous polyposis protein (APC)), immunomodulatory specific peptides, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY 2D), rab guard 1 (no choroidal disease), LCA5 (LCA-Le Beixi forest (lebericlin)), ornithine ketoacid aminotransferase (Gyrate atropy), retinal cleavage 1 (X-linked retinal cleavage), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinal pigment degeneration), DFNB1 (gap junction protein 26 deafness), ACHM 2, 3 and 4 (total color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin a, GM2-AP, NPC1, VPC2, sphingosine activator protein, one or more zinc finger nucleases for genome editing, or one or more donor sequences for use as repair templates for genome editing.

In certain embodiments, the heterologous polynucleotide encodes Erythropoietin (EPO) for the treatment of anemia; interferon alpha, interferon-beta and interferon-gamma for the treatment of various immune disorders, viral infections and cancers; interleukins (IL), including any of IL-1 to IL-36, and the corresponding receptors, for the treatment of various inflammatory diseases or immunodeficiency; chemokines, including chemokine (C-X-C motif) ligand 5 (CXCL 5), for use in the treatment of immune disorders; granulocyte-colony stimulating factor (G-CSF) for use in the treatment of immune disorders such as Crohn's disease; granulocyte-macrophage colony stimulating factor (GM-CSF) for use in the treatment of various human inflammatory diseases; macrophage colony stimulating factor (M-CSF) for use in the treatment of various human inflammatory diseases; keratinocyte Growth Factor (KGF) for the treatment of epithelial tissue injury; chemokines such as monocyte chemoattractant protein-1 (MCP-1) for the treatment of recurrent abortion, HIV-related complications and insulin resistance; tumor Necrosis Factor (TNF) and receptors for the treatment of various immune disorders; alpha 1-antitrypsin for use in the treatment of emphysema or Chronic Obstructive Pulmonary Disease (COPD); alpha-L-iduronidase for the treatment of mucopolysaccharidosis I (MPS I); ornithine Transcarbamylase (OTC) for the treatment of OTC deficiency; phenylalanine hydroxylase (PAH) or Phenylalanine Ammonia Lyase (PAL) for use in the treatment of Phenylketonuria (PKU); lipoprotein lipase for treating lipoprotein lipase deficiency; an apolipoprotein for use in the treatment of apolipoprotein (Apo) a-I deficiency; low density lipoprotein receptor (LDL-R) for the treatment of Familial Hypercholesterolemia (FH); albumin for use in the treatment of hypoalbuminemia; lecithin Cholesterol Acyltransferase (LCAT); carbamoyl synthase I; argininosuccinate synthetase; argininosuccinate lyase; arginase; fumarylacetoacetic acid hydrolase; porphobilinogen deaminase; cystathionine beta-synthase for use in the treatment of homocystinuria; branched-chain keto acid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl CoA carboxylase; methylmalonyl-CoA mutase; glutaryl CoA dehydrogenase; insulin; a pyruvate carboxylase; liver phosphorylase; phosphorylase kinase; glycine decarboxylase; h-protein; t-protein; cystic Fibrosis Transmembrane Regulator (CFTR); ATP-binding cassette, subfamily a (ABC 1), member 4 (ABCA 4), for use in the treatment of stargardt disease (Stargardt disease); or dystrophin (dystraphin).

In certain embodiments, the protein encoded by the heterologous polynucleotide comprises a gene editing nuclease. In certain embodiments, the gene editing nuclease comprises a Zinc Finger Nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). In certain embodiments, the gene editing nuclease comprises a functional type II CRISPR-Cas9.

The terms "polypeptide", "protein" and "peptide" are used interchangeably herein. "polypeptide", "protein" and "peptide" encoded by a "polynucleotide sequence" include the same full-length native sequence as a naturally occurring protein, as well as functional sequences, modified forms or sequence variants, so long as the subsequence, modified form or variant retains some degree of functionality of the native full-length protein. In the present invention, such polypeptides, proteins and peptides encoded by the polynucleotide sequences may, but are not required to, be identical to endogenous proteins that are defective in the mammal being treated, or that are under-expressed, or that are lacking.

In certain embodiments, the heterologous polynucleotide encodes an inhibitory nucleic acid selected from the group consisting of siRNA, antisense molecule, miRNA, RNAi, ribozyme, and shRNA.

In certain embodiments, the inhibitory nucleic acid binding gene, transcript of a gene or transcript of a gene associated with a polynucleotide repeat disease is selected from Huntingtin (HTT) gene, gene associated with dentate nuclear pallidolulin atrophy (atropin 1, atn 1)), androgen receptor on the X chromosome in spinobulnar muscular atrophy, human spinocerebellar Ataxin (Ataxin) -1, -2, -3 and-7, cav 2.1P/Q voltage-dependent calcium channel (CACNA 1A), TATA-binding protein, spinocerebellar Ataxin 8 reverse chain (ATXN 8 OS), serine/threonine-protein phosphatase 2a55kDa regulatory subunit bβ isoform in spinocerebellar ataxia (1, 2, 3, 6, 7, 8, 12 types), FMR1 in fragile X syndrome (fmx 1), fragile X-related tremor/co-fragile X syndrome (fragile X1), fragile X1 or fragile X2 members in fmx 2 family of intellectual disability (fmx 1); myotonic protein kinase (MT-PK) in myotonic muscular dystrophy; ataxin in friedreich ataxia (Frataxin); mutants of superoxide dismutase 1 (SOD 1) in amyotrophic lateral sclerosis; genes involved in the pathogenesis of parkinson's disease and/or alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin 9 (proprotein convertase subtilisin/kexin type 9, PCSK9), hypercholesterolemia; trans-activating factor of HIV Tat, human immunodeficiency virus transcription gene, in HIV infection; HIV TAR, human immunodeficiency virus transactivator response element genes, in HIV infection; the C-C chemokine receptor (CCR 5) in HIV; the Rous Sarcoma Virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis c virus infection; p53, acute kidney injury or delayed graft function kidney transplantation or acute kidney failure from kidney injury; protein kinase N3 (PKN 3) in advanced recurrent or metastatic solid malignancies; LMP2, LMP2 is also known as proteasome subunit beta-9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-8 (PSMB 8), metastatic melanoma; MECL1, also known as proteasome subunit β -10 (PSMB 10), metastatic melanoma; vascular Endothelial Growth Factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis-inhibiting factor B-cell CLL/lymphoma (BCL-2) in chronic myelogenous leukemia; ribonucleotide reductase M2 (RRM 2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK 1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT 1) in hepatitis c infection, β -catenin in familial adenomatous polyposis; beta 2 adrenergic receptors, glaucoma; RTP801/Redd1, also known as DAN damage inducing transcript 4 protein, in Diabetic Macular Edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR 1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6AN17K muteins in congenital pachyrhizus; influenza a virus genome/gene sequence in influenza infection; severe Acute Respiratory Syndrome (SARS) coronavirus genome/gene sequence in SARS infection; respiratory syncytial virus genome/gene sequence in respiratory syncytial virus infection; ebola filovirus genome/gene sequences in ebola virus infection; the hepatitis b and c viral genome/gene sequences in hepatitis b and c viral infections; herpes Simplex Virus (HSV) genome/gene sequence in HSV infection, coxsackievirus B3 genome/gene sequence in coxsackievirus B3 infection; silencing of pathogenic alleles of genes (allele-specific silencing), such as torsin a, TOR1A in primary dystonia, pan-class I (pan-class I) and HLA-alleles specific in transplantation; and mutant rhodopsin gene (RHO) in autosomal dominant inherited retinal pigment degeneration (adRP).

Immune cell modulators

In certain embodiments, the invention relates to a method of producing a therapeutically effective non-viral gene therapy in a subject comprising administering to the subject at least one immune cell modulating agent, e.g., at least one phagocytic depleting agent, or at least one immunosuppressant.

As used herein, the term "phagocytic depleting agent" refers to any agent that depletes or destroys phagocytes of a subject and/or interferes with one or more phagocytic functions. The phagocytic depleting agent may target any phagocyte. Phagocytes (Phagocyte), also referred to herein as phagocytes (Phagocyte cells), phagocytes (Phagocyte cells), or phagocytes immune cells, including, for example, macrophages, monocytes, neutrophils, and dendritic cells. Langerhans cells (Langerhans cells) are dendritic cells found in the skin. Mast cells found in many tissues including, for example, the lung or skin, can also act as phagocytes.

As used herein, the term "monocyte and/or macrophage depleting agent" refers to any agent that depletes or destroys monocytes and/or macrophages and/or interferes with one or more monocyte and/or macrophage functions in a subject. The monocyte and/or macrophage depleting agent may target any monocyte and/or macrophage. Macrophages are single nuclear phagocytes and are differentiated monocytes. Macrophages are called different names in different tissues. Examples of tissue-specific or resident macrophages include, for example, kupffer cells in the liver, intestinal macrophages in the intestine, microglia cells in the brain, alveolar macrophages in the lung, resident kidney macrophages, skin macrophages, red marrow macrophages in the spleen, and osteoclasts in the bone. Examples of monocyte and/or macrophage depleting agents include, for example, agents that target phagocytic immune cell markers, e.g., CD115 inhibitors, including but not limited to anti-CD 115 antibodies or CD115 small molecule inhibitors; f4/80 inhibitors, including but not limited to anti-F4/80 antibodies or F4/80 small molecule inhibitors; CD68 inhibitors, including but not limited to anti-CD 68 antibodies or CD68 small molecule inhibitors; CD11b inhibitors, including but not limited to anti-CD 11b antibodies or CD11b small molecule inhibitors; the chemotherapeutic agent Trabectedin (Trabectedin); fat milk (intra fat); empty liposomes; and bisphosphonates including, but not limited to, chlorophosphonate. In certain embodiments, the monocyte and/or macrophage depleting agent is not a chlorophosphonate. In certain embodiments, the chlorophosphonate is used with at least one additional monocyte and/or macrophage depleting agent in the methods of the present invention.

As used herein, the term "neutrophil depleting agent" refers to any agent that depletes or disrupts neutrophils and/or interferes with one or more neutrophil functions in a subject. The neutrophil depleting agent may target any neutrophil. Examples of neutrophil depleting agents include, for example, agents that target phagocytic immune cell markers, e.g., ly6G inhibitors, including but not limited to anti-Ly 6G antibodies or Ly6G small molecule inhibitors; CD177 inhibitors, including but not limited to anti-CD 177 antibodies or CD177 small molecule inhibitors; CD14 inhibitors, including but not limited to anti-CD 14 antibodies or CD14 small molecule inhibitors; CD15 inhibitors, including but not limited to anti-CD 15 antibodies or CD15 small molecule inhibitors; CD11b inhibitors, including but not limited to anti-CD 11b antibodies or CD11b small molecule inhibitors; CD16 inhibitors, including but not limited to anti-CD 16 antibodies or CD16 small molecule inhibitors; CD32 inhibitors, including but not limited to anti-CD 32 antibodies or CD32 small molecule inhibitors; CD33 inhibitors, including but not limited to anti-CD 33 antibodies or CD33 small molecule inhibitors; CD44 inhibitors, including but not limited to anti-CD 44 antibodies or CD44 small molecule inhibitors; CD45 inhibitors, including but not limited to anti-CD 45 antibodies or CD45 small molecule inhibitors; CD66b inhibitors, including but not limited to anti-CD 66b antibodies or CD66b small molecule inhibitors; CD18, or inhibitors, including but not limited to anti-CD 18 antibodies or CD18 small molecule inhibitors; CD62L inhibitors, including but not limited to anti-CD 62L antibodies or CD62L small molecule inhibitors; and Gr-1 inhibitors, including but not limited to anti-Gr-1 antibodies or Gr-1 small molecule inhibitors.

As used herein, the term "dendritic cell depleting agent" refers to any agent that depletes or destroys dendritic cells of a subject and/or interferes with one or more dendritic functions. The dendritic cell depleting agent can target any dendritic cell. Examples of dendritic cell depleting agents include, for example, agents that target phagocytic immune cell markers, e.g., PDCA1 inhibitors, including but not limited to anti-PDCA 1 antibodies or small molecule PDCA1 inhibitors; and CD11c inhibitors, including but not limited to anti-CD 11c antibodies or CD11c small molecule inhibitors.

As used herein, the term "inhibitor" refers to any compound capable of down-regulating, reducing, decreasing, inhibiting or inactivating the amount and/or activity of a target protein. Inhibitors may be proteins, oligopeptides and polypeptides, nucleic acids, genes or chemical molecules. Suitable protein inhibitors may be, for example, monoclonal or polyclonal antibodies that bind to the target protein.

Any suitable CD115 inhibitor, including those known to those skilled in the art, may be used in the present invention in view of this disclosure. Examples of small molecule inhibitors of CD115 include, for example, pexidastinib (PLX-3397), BLZ-945, li Nifa Ni (Linifanib) (ABT-869), JNJ-28312141 (Johnson & Johnson), JNJ-40346527 (Johnson & Johnson), PLX7486 (Plexxikon), and ARRY-382 (Array BioPharma).

Any suitable anti-CD 115 antibodies, including those known to those of skill in the art, may be used in the present invention in view of the present disclosure. Examples of commercial anti-CD 115 antibodies include, for example, AFS98 (Invitrogen or Biocell), 12-3A3-1B10 (Invitrogen), 6C7 (Bioss), carbitol (FPA 008), 25949-1-AP (Proteintech), 1G4 (Abnova), 3G12 (Abnova), 604B 5E 11 (Invitrogen), E Mi Tuozhu mab (RG-7155; roche), AMG 820 (Amgen), IMC-CS4, and ROS8G11 (Invitrogen). In certain embodiments, the antibody or antigen binding fragment is AFS98 (e.g., bioCell BE 0213), see also oncogene.1995;11 (12):2469-2476.

Any suitable Ly6G inhibitor, including those known to those skilled in the art, may be used in the present invention in view of this disclosure.

Any suitable anti-Ly 6G antibody, including those known to those skilled in the art, may be used in the present invention in view of the present disclosure. Examples of commercial anti-Ly 6G antibodies include, for example, 1A8 (Biocell BP 0075-1) and RB6-8C5 (ab 25377).

In view of the present disclosure, any suitable anti-CD 177 antibody or CD177 small molecule inhibitor, including those known to those skilled in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 14 antibody or CD14 small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 15 antibody or CD15 small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 11b antibody or CD11b small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 16 antibody or CD16 small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 32 antibody or CD32 small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 33 antibody or CD33 small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 44 antibody or CD44 small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 45 antibody or CD45 small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 66b antibody or CD66b small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 18 antibody or CD18 small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention. In view of the present disclosure, any suitable anti-CD 62L antibody or CD62L small molecule inhibitor, including those known to those of skill in the art, may be used in the present invention.

Fat milk and empty liposomes have been shown to interfere with one or more functions of monocytes and/or macrophages. See, e.g., liu et al, biochem Biophys acta.2013 Jun;1830 (6) 3447-53 and Saunders et al, nano Lett.2020 Jun 10;20 (6):4264-4269. Pretreatment with fat milk or empty liposomes can effectively saturate monocytes/macrophages and prevent phagocytosis of non-viral therapeutic agents. Any suitable fat emulsion or empty liposome known to those skilled in the art may be used in the present invention in view of the present disclosure. Examples of fat emulsions and empty liposomes include, for example, I141-100ML (Sigma Aldrich), 2B6063 (Baxter), and Liu et al, biochem Biophys acta.2013 Jun;1830 (6) 3447-53 and Saunders et al, nano Lett.2020 Jun 10;20 (6) those described in 4264-4269.

Any suitable bisphosphonate known to those skilled in the art may be used in the present invention in view of this disclosure. Examples of bisphosphonates include, for example, clodronate, pamidronate, ibandronate, alendronate, and zoledronate.

Other examples of "phagocytic depletion agents" include, for example, palbociclib (palbociclib) Pfizer), cromolyn sodium (>Bausch&Lomb), they are known to inhibit mast cells.

As used herein, the term "immunosuppressant" refers to any compound capable of slowing or stopping the activity of the subject's immune system. Examples of immunosuppressants include, but are not limited to, calcium-dependent phosphatase inhibitors such as, but not limited to, cyclosporin, ISA (TX) 247, tacrolimus (tacrolimus) or calcium-dependent phosphatase, targets of rapamycin such as, but not limited to, sirolimus (sirolimus), everolimus (everolimus), FK778 or TAFA-93, interleukin-2 alpha-chain blockers such as, but not limited to, basiliximab and daclizumab, inhibitors of inosine monophosphate dehydrogenase such as, but not limited to, mycophenolate mofetil (mycophenolate mofetil), inhibitors of dihydrofolate reductase such as, but not limited to, methotrexate, immunosuppressive antimetabolites such as, but not limited to, azathioprine, JAK inhibitors such as, but not limited to, ruxolitinib), cytokine inhibitors such as, but not limited to, anti-cytokine antibodies such as, but not limited to, seluximab (sibamab), cGAS, but not limited to, H151-steroids or steroids.

As used herein, the term "steroid" is used to include corticosteroids and glucocorticosteroids. In certain embodiments, the steroid may be a corticosteroid. Any suitable corticosteroid known to those skilled in the art may be used in the present invention in view of this disclosure. As used herein, the term "steroid" refers to a chemical comprising three cyclohexane rings and one cyclopentane ring. These rings are arranged to form a tetracyclic pentaphenanthrene (cycloparaffin), a steroid (gonane). As used herein, the term "corticosteroid" refers to a class of steroid hormones produced or synthetically produced in the adrenal cortex. Corticosteroids are involved in a wide range of physiological systems such as regulation of stress responses, immune responses and inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels (blood electrolyte level) and behavior. Corticosteroids are generally classified into four classes based on chemical structure. Group a corticosteroids (short to medium acting glucocorticoids) include hydrocortisone, hydrocortisone acetate, ticortisone pivalate (tixocortol pivalate), prednisolone, methylprednisolone, and prednisone. Group B corticosteroids include triamcinolone acetonide, triamcinolone Long Chun (triamcinolone alcohol), mometasone (mometasone), ambroxide, budesonide, fluocinolone acetonide, fluocinonide (fluocinolone acetonide), and halcinonide. Group C corticosteroids include betamethasone, betamethasone sodium phosphate, dexamethasone sodium phosphate, and fludrolone. Group D corticosteroids include hydrocortisone-17-butyrate, hydrocortisone-17-valerate, beclomethasone dipropionate (aclometasone dipropionate), betamethasone valerate, betamethasone dipropionate, prednisone, clobetasone-17-butyrate, clobetasol-17-propionate, fluacolone caproate (fluocortolone caproate), fluacolone valerate (fluocortolone pivalate), and fluprednisodine acetate. Non-limiting examples of corticosteroids Examples include aldosterone (aldosterone), beclomethasone dipropionate, betamethasone (betamethasone), betamethasone-21-phosphate disodium, betamethasone valerate, budesonide, clobetasol propionate, clobetasol butyrate, clobetasol pivalate, cortisol, corticosterone (cortisteron), cortisone, deflazacort, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone acetate, dihydroxycortisone (dihydroxycortisone), fluocinolone acetonide Fluorometasone, flunisolide acetate (flucionolone acetonide), fluticasone furoate, fluticasone propionate, halpmetasone, halometasone, hydrocortisone acetate, hydrocortisone succinate, 16 alpha-hydroxy prednisolone, isophorone acetate, meflosone, methylprednisolone, desonide (prednisolone), prednisolide (prednisorate), prednisolone acetate, prednisolone sodium succinate, prednisone, triamcinolone, and triamcinolone diacetate. As used herein, the term "corticosteroid" may include, but is not limited to, corticosteroids of the following generic and brand names: cortisone (cortoine) ^TM ACETATE ^TM 、ADRESON ^TM 、ALTESONA ^TM 、CORTELANT ^TM 、CORTISTAB ^TM 、CORTISYL ^TM 、CORTOGEN ^TM 、CORTONE ^TM 、SCHEROSON ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the dexamethasone-ORAL (Decadron ORAL) ^TM 、DEXAMETH ^TM 、DEXONE ^TM 、HEXADROL-ORAL ^TM 、DEXAMETHASONE ^TM INTENSOL ^TM 、DEXONE 0.5 ^TM 、DEXONE 0.75 ^TM 、DEXONE 1.5 ^TM 、DEXONE 4 ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the Hydrocortisone-oral (CORTEF) ^TM 、HYDROCORTONE ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the Hydrocortisone cyclopentanepropionate (CORTEF ORAL SUSPENSION) ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the Methylprednisolone ORAL (MEDROL-ORAL) ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the Prednisolone oral (PRELONE) ^TM 、DELTA-CORTEF ^TM 、PEDIAPRED ^TM 、ADNISOLONE ^TM 、CORTALONE ^TM 、DELTACORTRIL ^TM 、DELTASOLONE ^TM 、DELTASTAB ^TM 、DI-ADRESON F ^TM 、ENCORTOLONE ^TM 、HYDROCORTANCYL ^TM 、MEDISOLONE ^TM 、METICORTELONE ^TM 、OPREDSONE ^TM 、PANAAFCORTELONE ^TM 、PRECORTISYL ^TM 、PRENISOLONA ^TM 、SCHERISOLONA ^TM 、SCHERISOLONE ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the Prednisone (DeltaSONE) ^TM 、LIQUID PRED ^TM 、METICORTENT ^TM 、ORASONE 1 ^TM 、ORASONE 5 ^TM 、ORASONE 10 ^TM 、ORASONE 20 ^TM 、ORASONE 50 ^TM 、PREDNICEN-M ^TM 、PREDNISONE INTENSOL ^TM 、STERAPRED ^TM 、STERAPRED DS ^TM 、ADASONE ^TM 、CARTANCYL ^TM 、COLISONE ^TM 、CORDROL ^TM 、CORTAN ^TM 、DACORTIN ^TM 、DECORTIN ^TM 、DECORTISYL ^TM 、DELCORTIN ^TM 、DELLACORT ^TM 、DELTADOME ^TM 、DELTACORTENE ^TM 、DELTISONA ^TM 、DIADRESON ^TM 、ECONOSONE ^TM 、ENCORTON ^TM 、FERNISONE ^TM 、NISONA ^TM 、NOVOPREDNISONE ^TM 、PANAFCORT ^TM 、PANASOL ^TM 、PARACORT ^TM 、PARMENISON ^TM 、PEHACORT ^TM 、PREDELTIN ^TM 、PREDNICORT ^TM 、PREDNICOT ^TM 、PREDNIDIB ^TM 、PREDNIMENT ^TM 、RECTODELT ^TM 、ULTRACORTEN ^TM 、WINPRED ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the Oral triamcinolone (triamcinolone acetonide) (kenarort) ^TM 、ARISTOCORT ^TM 、ATOLONE ^TM 、SHOLOG A ^TM 、TRAMACORT-D ^TM 、TRI-MED ^TM 、TRIAMCOT ^TM 、TRISTOPLEX ^TM 、TRYLONE D ^TM 、U-TRI-LONE ^TM ). In certain embodiments, the corticosteroid may be dexamethasone, prednisone, prednisolone, triamcinolone, clobetasol propionate, betamethasone valerate, betamethasone dipropionate, or mometasone furoate. Methods for synthesizing steroids and corticosteroids are well known in the art, and such compounds are also commercially available, e.g., dexamethasone(catalog number D4902, sigma-Aldrich; st. Louis, mo.) and prednisone (catalog number P6254, sigma-Aldrich; st. Louis, mo.).

Corticosteroids, such as dexamethasone, may be delivered as free dexamethasone. Alternatively, a corticosteroid, e.g., dexamethasone, can be delivered by LNP, either as a separate LNP composition or as part of the same LNP composition as a therapeutic transgene.

Therapeutic compositions

In certain embodiments, the invention relates to a therapeutic composition comprising a phagocytic depleting agent of the invention for the treatment of a disease treated by gene therapy in a patient in need thereof using a pharmaceutical composition of the invention, the pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier.

Any therapeutic agent or pharmaceutical composition of the invention may be combined with a pharmaceutically acceptable excipient, and optionally a slow release matrix (matrix), such as a biodegradable polymer, to form a therapeutic composition.

By "pharmaceutically" or "pharmaceutically acceptable" is meant molecular entities and compositions which, when administered to a mammal, particularly a human, do not produce adverse (overtse), allergic or other untoward) reactions, under appropriate circumstances. Pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation auxiliary.

The form, route of administration, dosage and regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the condition, the age, weight and sex of the patient, etc.

The pharmaceutical compositions of the present invention may be formulated for topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, or subcutaneous administration, and the like.

The pharmaceutical compositions of the invention may contain a carrier that is pharmaceutically acceptable for injectable formulations. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts), or dry, in particular freeze-dried, compositions, which, as the case may be, allow the composition of injectable solutions upon addition of sterile water or physiological saline. The dosage for administration may be adjusted as a function of various parameters, in particular as a function of the mode of administration used, of the associated pathology, or of the desired treatment time.

Reference to a pharmaceutically acceptable salt means that the salt is suitable for administration to a mammal, preferably a human, in the amounts provided.

In addition, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; time release capsules; and any other form that may be presently used.

The pharmaceutical compositions of the present invention may comprise further therapeutically active agents.

The pharmaceutical composition comprising the non-viral delivery nanoparticle and the non-viral vector comprising the therapeutic transgene operably linked to the promoter may be administered to a subject at any suitable dose. For example, a suitable dose may be from about 0.01mg/ml to about 10mg/ml of non-viral vector per kg body weight of the subject.

Antibodies targeting phagocytic immune cell markers may be administered to a subject at any suitable dose. For example, a suitable dose may be from about 0.01mg/kg to about 5mg/kg of subject body weight, wherein the dose is administered in 1-10 total injections.

The anti-CD 115 antibodies may be administered to the subject in any suitable dosage. For example, a suitable dose may be from about 0.01mg/kg to about 5mg/kg of subject body weight, wherein the dose is administered in 1-10 total injections. In certain embodiments, the dosage may be from about 0.01mg/kg to about 0.1mg/kg, about 0.1mg/kg to about 0.2mg/kg, about 0.2mg/kg to about 0.3mg/kg, about 0.3mg/kg to about 0.4mg/kg, about 0.4mg/kg to about 0.5mg/kg, about 0.5mg/kg to about 0.6mg/kg, about 0.6mg/kg to about 0.7mg/kg, about 0.7mg/kg to about 0.8mg/kg, about 0.8mg/kg to about 0.9mg/kg, about 0.9mg/kg to about 1mg/kg, about 1mg/kg to about 1.5mg/kg, about 1.5mg/kg to about 2mg/kg, about 2.5mg/kg to about 3mg/kg, about 3.7 mg/kg to about 3.8 mg/kg, about 0.8mg/kg to about 1.5mg/kg, about 4mg/kg to about 4.5 mg/kg. In certain embodiments, the dose is less than any of the following doses (in mg/kg): 5. 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or 0.02. In certain embodiments, the dose is greater than any of the following doses (in mg/kg): 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 5, 4.6, 4.7, 4.8 or 4.9. That is, the dosage may be of upper limit 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or 0.02, and the independently selected lower limit is a dose of any of the lower limits (mg/ml) of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4.4, 4.5, 4.6, 4.7, 4.8 or 4.9.

The anti-Ly 6G, anti-CD 177, anti-CD 14, anti-CD 15, anti-CD 11b, anti-CD 16, anti-CD 32, anti-CD 33, anti-CD 44, anti-CD 45, anti-CD 66b, anti-CD 18, or anti-CD 62L antibody may be administered to a subject in any suitable dosage. For example, a suitable dose may be from about 0.01mg/kg to about 5mg/kg of subject body weight, wherein the dose is administered in 1-10 total injections. In certain embodiments, the dosage may be from about 0.01mg/kg to about 0.1mg/kg, about 0.1mg/kg to about 0.2mg/kg, about 0.2mg/kg to about 0.3mg/kg, about 0.3mg/kg to about 0.4mg/kg, about 0.4mg/kg to about 0.5mg/kg, about 0.5mg/kg to about 0.6mg/kg, about 0.6mg/kg to about 0.7mg/kg, about 0.7mg/kg to about 0.8mg/kg, about 0.8mg/kg to about 0.9mg/kg, about 0.9mg/kg to about 1mg/kg, about 1mg/kg to about 1.5mg/kg, about 1.5mg/kg to about 2mg/kg, about 2.5mg/kg to about 3mg/kg, about 3.7 mg/kg to about 3.8 mg/kg, about 0.8mg/kg to about 1.5mg/kg, about 4mg/kg to about 4.5 mg/kg. In certain embodiments, the dose is less than any of the following doses (in mg/kg): 5. 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or 0.02. In certain embodiments, the dose is greater than any of the following doses (in mg/kg): 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 5, 4.6, 4.7, 4.8 or 4.9. That is, the dosage may be of upper limit 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or 0.02, and the independently selected lower limit is a dose of any of the lower limits (mg/ml) of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4.4, 4.5, 4.6, 4.7, 4.8 or 4.9.

The small molecule inhibitors that target phagocytic immune cell markers may be administered to a subject at any suitable dose. For example, a suitable dosage may be from about 0.1mg/kg to about 15mg/kg of subject body weight. In certain embodiments, the dosage may be from about 0.1mg/kg to about 1mg/kg, about 1mg/kg to about 2mg/kg, about 2mg/kg to about 3mg/kg, about 3mg/kg to about 4mg/kg, about 4mg/kg to about 5mg/kg, about 5mg/kg to about 6mg/kg, about 6mg/kg to about 7mg/kg, about 7mg/kg to about 8mg/kg, about 8mg/kg to about 9mg/kg, about 9mg/kg to about 10mg/kg, about 10mg/kg to about 11mg/kg, about 11mg/kg to about 12mg/kg, about 12mg/kg to about 13mg/kg, about 13mg/kg to about 14mg/kg, about 14mg/kg to about 15mg/kg. In certain embodiments, the dose is less than any of the following doses (in mg/kg): 15. 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2. In certain embodiments, the dose is greater than any of the following doses (in mg/kg): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. That is, the dose may be any of the dose ranges (in mg/ml) having an upper limit of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2, and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

CD115 inhibitors such as pexidasatinib may be administered to a subject in any suitable dose. For example, a suitable dosage may be from about 0.1mg/kg to about 15mg/g of subject body weight. In certain embodiments, the dosage may be from about 0.1mg/kg to about 1mg/kg, about 1mg/kg to about 2mg/kg, about 2mg/kg to about 3mg/kg, about 3mg/kg to about 4mg/kg, about 4mg/kg to about 5mg/kg, about 5mg/kg to about 6mg/kg, about 6mg/kg to about 7mg/kg, about 7mg/kg to about 8mg/kg, about 8mg/kg to about 9mg/kg, about 9mg/kg to about 10mg/kg, about 10mg/kg to about 11mg/kg, about 11mg/kg to about 12mg/kg, about 12mg/kg to about 13mg/kg, about 13mg/kg to about 14mg/kg, about 14mg/kg to about 15mg/kg. In certain embodiments, the dose is less than any of the following doses (in mg/kg): 15. 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2. In certain embodiments, the dose is greater than any of the following doses (in mg/kg): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. That is, the dose may be any of the dose ranges (in mg/ml) having an upper limit of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2, and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

Bisphosphonates, e.g., clodronate, may be administered to a subject in any suitable dosage. For example, a suitable dosage may be from about 0.1mg/kg to about 15mg/kg of subject body weight. In certain embodiments, the dosage may be from about 0.1mg/kg to about 1mg/kg, about 1mg/kg to about 2mg/kg, about 2mg/kg to about 3mg/kg, about 3mg/kg to about 4mg/kg, about 4mg/kg to about 5mg/kg, about 5mg/kg to about 6mg/kg, about 6mg/kg to about 7mg/kg, about 7mg/kg to about 8mg/kg, about 8mg/kg to about 9mg/kg, about 9mg/kg to about 10mg/kg, about 10mg/kg to about 11mg/kg, about 11mg/kg to about 12mg/kg, about 12mg/kg to about 13mg/kg, about 13mg/kg to about 14mg/kg, about 14mg/kg to about 15mg/kg. In certain embodiments, the dose is less than any of the following doses (in mg/kg): 15. 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2. In certain embodiments, the dose is greater than any of the following doses (in mg/kg): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. That is, the dose may be any of the dose ranges (in mg/ml) having an upper limit of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2, and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

The corticosteroid, e.g., dexamethasone, may be administered to the subject at any suitable dose. For example, a suitable dosage may be from about 0.1mg/kg to about 15mg/kg of subject body weight. In certain embodiments, the dosage may be from about 0.1mg/kg to about 1mg/kg, about 1mg/kg to about 2mg/kg, about 2mg/kg to about 3mg/kg, about 3mg/kg to about 4mg/kg, about 4mg/kg to about 5mg/kg, about 5mg/kg to about 6mg/kg, about 6mg/kg to about 7mg/kg, about 7mg/kg to about 8mg/kg, about 8mg/kg to about 9mg/kg, about 9mg/kg to about 10mg/kg, about 10mg/kg to about 11mg/kg, about 11mg/kg to about 12mg/kg, about 12mg/kg to about 13mg/kg, about 13mg/kg to about 14mg/kg, about 14mg/kg to about 15mg/kg. In certain embodiments, the dose is less than any of the following doses (in mg/kg): 15. 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2. In certain embodiments, the dose is greater than any of the following doses (in mg/kg): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. That is, the dose may be any of the dose ranges (in mg/ml) having an upper limit of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2, and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

Method

In certain embodiments, the invention relates to a method of delivering a transgene to a subject in need thereof, the method comprising (a) administering to the subject a monocyte and/or macrophage depleting agent; and (b) administering to the subject a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier.

As used herein, the term "disease treated by gene therapy using a non-viral vector" refers to a disease in which a polynucleotide (encoding at least one polypeptide or inhibitory nucleic acid) is delivered as a drug into cells of a patient to treat the disease (gene therapy).

In certain embodiments, the vector encodes at least one specific polypeptide or inhibitory nucleic acid useful for treating a disease. In certain embodiments, the vector encodes/comprises a therapeutic polynucleotide suitable for treating a disease.

In certain embodiments, the vector encodes/comprises a therapeutic polynucleotide suitable for treating a disease using gene therapy.

As used herein, the terms "patient" and "subject" interchangeably refer to animals, typically mammals, such as rodents, felines, canines, and primates. In particular, the subject or patient of the invention is a human.

As used herein, the term "treatment" or "treatment" refers to prophylactic or preventative treatments, such as gene therapy and curative or disease modifying treatments, including treatment of subjects at risk of, or suspected of having, an infectious disease, as well as subjects suffering from, or diagnosed with, a disease or medical condition, and includes inhibition of clinical recurrence. The treatment may be administered to a subject suffering from, or ultimately having available a medical condition, to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the condition or recurrent condition, or in order to prolong survival of the subject beyond that expected in the absence of such treatment.

The methods of the invention may be performed in any suitable order unless otherwise indicated herein. In certain embodiments, the method can include first (a) administering a phagocytic depleting agent to a subject, and then (b) administering to the subject a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier. In certain embodiments, the administering of the pharmaceutical composition to the subject (b) occurs between about 1 minute and about 1 year after the administration of the phagocytic depleting agent to the subject (a) comprises a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier. In certain embodiments, administering to the subject (a) a phagocytic depleting agent and (b) a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier are performed at about the same time.

In certain embodiments, the method can include first (a) administering to the subject a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier, and then (b) administering to the subject a phagocytic depleting agent. In certain embodiments, (a) administering to the subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier, followed by (b) administering to the subject a phagocytic depleting agent between about 1 minute and about 1 year. In certain embodiments, administering to the subject a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier, and (b) administering to the subject a phagocytic depleting agent is performed at about the same time.

In certain embodiments, the phagocytic depleting agent is administered between about 1 minute and about 1 hour, 2 hours, 3 hours, or 4 hours prior to administration of the non-viral vector comprising the transgene and the pharmaceutically acceptable carrier.

In certain embodiments, the non-viral vector comprising the transgene and the pharmaceutically acceptable carrier are administered between about 1 minute and about 1 hour, 2 hours, 3 hours, or 4 hours prior to administration of the phagocyte depleting agent.

In certain embodiments, the phagocytic depleting agent is administered at least one day prior to administration of the non-viral vector, optionally no more than 1 year prior to administration of the non-viral vector, e.g., no more than 52 weeks, 51 weeks, 50 weeks, 49 weeks, 48 weeks, 47 weeks, 46 weeks, 45 weeks, 44 weeks, 43 weeks, 42 weeks, 41 weeks, 40 weeks, 39 weeks, 38 weeks, 37 weeks, 36 weeks, 35 weeks, 34 weeks, 33 weeks, 32 weeks, 31 weeks, 30 weeks, 29 weeks, 28 weeks, 27 weeks, 26 weeks, 25 weeks, 24 weeks, 23 weeks, 22 weeks, 21 weeks, 20 weeks, 19 weeks, 18 weeks, 17 weeks, 16 weeks, 15 weeks, 14 weeks, 13 weeks, 12 weeks, 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 day, 6 days, 5 days, 4 days, 3 days, or 2 days prior to administration of the non-viral vector.

The non-viral vector comprising the heterologous polynucleotide may be administered to the subject any number of times. For example, a non-viral vector comprising a heterologous polynucleotide may be administered to a subject 1, 2-15 times. In certain embodiments, the non-viral vector is administered multiple times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The interval between each may vary, for example, every 1 hour, 6 hours, 24 hours, 1-7 days, or 2-10 weeks, or a combination thereof.

The non-viral vector comprising the heterologous polynucleotide may be administered to the subject periodically, such as for consecutive days, or alternatively for days, or aperiodically, over any period of time. In certain embodiments, the non-viral vector comprising the heterologous polynucleotide is administered about 1-52 weeks after administration of the immune cell modulating agent.

The phagocytic depleting agent may be administered to the subject any number of times. For example, a non-viral vector comprising a heterologous polynucleotide may be administered to a subject 1, 2-5, 2-10, 2-15 times per administration of a phagocytic depleting agent.

The anti-CD 115 antibody may be administered to the subject any number of times. For example, the anti-CD 115 antibody may be administered 1, 2-5, 2-10, 2-15 times to the subject per administration of a non-viral vector comprising a heterologous polynucleotide.

The anti-CD 115 antibody may be administered to the subject periodically over any period of time, such as for consecutive days, or alternating days, or aperiodically. In certain embodiments, the anti-CD 115 antibody is administered about 1-52 weeks prior to administration of the non-viral vector.

The small molecule CD115 inhibitor may be administered to the subject any number of times. For example, a small molecule CD115 inhibitor may be administered to a subject 1, 2-5, 2-10, 2-15 times per administration of a non-viral vector comprising a heterologous polynucleotide.

The small molecule CD115 inhibitor may be administered to the subject periodically over any period of time, such as for several consecutive days, or alternatively for several days, or aperiodically. In certain embodiments, the small molecule CD115 inhibitor is administered about 1-52 weeks prior to administration of the non-viral vector.

The anti-Ly 6G, anti-CD 177, anti-CD 14, anti-CD 15, anti-CD 11b, anti-CD 16, anti-CD 32, anti-CD 33, anti-CD 44, anti-CD 45, anti-CD 66b, anti-CD 18, or anti-CD 62L antibody may be administered to the subject any number of times. For example, an anti-Ly 6G, anti-CD 177, anti-CD 14, anti-CD 15, anti-CD 11b, anti-CD 16, anti-CD 32, anti-CD 33, anti-CD 44, anti-CD 45, anti-CD 66b, anti-CD 18, or anti-CD 62L antibody may be administered 1, 2-5, 2-10, 2-15 times to a subject per administration of a non-viral vector comprising a heterologous polynucleotide.

The anti-Ly 6G, anti-CD 177, anti-CD 14, anti-CD 15, anti-CD 11b, anti-CD 16, anti-CD 32, anti-CD 33, anti-CD 44, anti-CD 45, anti-CD 66b, anti-CD 18, or anti-CD 62L antibodies may be administered to the subject periodically, such as for consecutive days, or alternating days, or aperiodically, for any period of time. In certain embodiments, the anti-Ly 6G, anti-CD 177, anti-CD 14, anti-CD 15, anti-CD 11b, anti-CD 16, anti-CD 32, anti-CD 33, anti-CD 44, anti-CD 45, anti-CD 66b, anti-CD 18, or anti-CD 62L antibody is administered about 1-52 weeks prior to administration of the non-viral vector.

Bisphosphonates, e.g., chlorophosphonate, may be administered to a subject any number of times. For example, each administration of a non-viral vector comprising a heterologous polynucleotide may administer a bisphosphonate, e.g., clodronate, 1, 2-5, 2-10, 2-15 times to a subject.

Bisphosphonates, e.g., chlorophosphonate, may be administered to a subject periodically over any period of time, such as for several consecutive days, or alternatively for several days, or aperiodically. In certain embodiments, the bisphosphonate, e.g., the chlorophosphonate, is administered about 1-52 weeks prior to administration of the non-viral vector.

The subject may be administered a corticosteroid, e.g., dexamethasone, any number of times. For example, a corticosteroid, e.g., dexamethasone, can be administered 1, 2-5, 2-10, 2-15 times to a subject per administration of a non-viral vector comprising a heterologous polynucleotide.

The corticosteroid, e.g., dexamethasone, may be administered to the subject periodically over any period of time, such as for consecutive days, or alternating days, or aperiodically. In certain embodiments, the corticosteroid, e.g., dexamethasone, is administered about 1-52 weeks prior to administration of the non-viral vector.

The non-viral vectors comprising the heterologous polynucleotide may be administered alone or in combination. In certain embodiments, the non-viral vector is administered separately from at least one immune cell modulating agent, such as a phagocytic depleting agent, bisphosphonate, to the subject; and/or immunosuppressants. In certain embodiments, the non-viral vector is administered to the subject in combination with at least one immune cell modulating agent, such as a phagocytic depleting agent, bisphosphonate; and/or immunosuppressants. In certain embodiments, the bisphosphonate and/or immunosuppressant is included within a non-viral delivery particle, e.g., encapsulated within a lipid nanoparticle, a polymer nanoparticle, a protein-based nanoparticle, or a peptide cage, along with a non-viral vector.

In certain embodiments, the subject is administered one or more times at least one immune cell modulating agent, such as a phagocytic depleting agent, bisphosphonate, and/or a mixture of immunosuppressants. In certain embodiments, two or more immune cell modulators are administered to a subject one or more times.

In certain embodiments, administration of at least one immune cell modulating agent to a subject can reduce the dose of non-viral vector comprising a heterologous polynucleotide required to effectively treat the subject. In certain embodiments, the administration of at least one immune cell modulating agent to a subject may allow for the administration of increased doses of a non-viral vector comprising a heterologous polynucleotide.

The dosage may vary and depends on the type, onset, progression, severity, frequency, duration, or probability of the disease for which treatment is being indicated, the desired clinical endpoint, previous or concurrent treatments, the general health, age, sex, race, or immunocompetence of the subject, and other factors that will be understood by the skilled artisan. The dose, number, frequency, or duration may be proportionally increased or decreased as indicated by any adverse side effects, complications, or other risk factors of the treatment or therapy, as well as the status of the subject. The skilled artisan will appreciate factors that can affect the dosage and time required to provide an amount sufficient to provide a therapeutic or prophylactic benefit.

Dosages to achieve a therapeutic effect, for example, in mg dsDNA per kilogram body weight (mg/kg), will vary based on several factors, including, but not limited to: the route of administration, the level of expression of the heterologous polynucleotide required to achieve a therapeutic effect, the particular disease being treated, any host immune response to the non-viral vector, any host immune response to the heterologous polynucleotide or expression product, and the stability of the expressed protein, peptide or nucleic acid. One skilled in the art can determine the non-viral vector genome dosage range for treating a patient suffering from a particular disease or disorder based on the factors described above as well as other factors.

An "effective amount" or "sufficient amount" refers to an amount that provides a detectable response for any length of time (long or short term), desired or expected result, or benefit to any measurable or detectable extent or any length of time (e.g., minutes, hours, days, months, years or cure) of a subject, alone or in combination with one or more other compositions, treatments, regimens (protocols) or therapeutic regimen (therapeutic regimen) agents, in a single dose or multiple doses. Dosages of an "effective amount" or "sufficient amount" to treat (e.g., alleviate or provide a therapeutic benefit or improvement) are generally effective to provide a response to one or more or all of the adverse symptoms, consequences, or complications of the disease, to a measurable extent, but reducing, lowering, inhibiting, suppressing, limiting, or controlling the progression or exacerbation of the disease (e.g., caused by or associated with the disease) are satisfactory results.

In certain embodiments, the methods of the invention result in a therapeutically effective non-viral gene therapy in a subject in need thereof, wherein the therapeutically effective non-viral gene therapy is characterized by minimal or no expression of a therapeutic protein or nucleic acid and/or an undesired immune response induced by a non-viral vector.

In certain embodiments, "expression of a therapeutic protein or nucleic acid" refers to a sufficient level of protein or nucleic acid to cause a therapeutic effect, as discussed herein.

In certain embodiments, the methods of the invention result in expression of the transgene in the subject.

In certain embodiments, the methods of the invention may result in the expression or activity of a therapeutic protein at a therapeutically effective level.

In certain embodiments, the methods of the invention can result in expression or activity of a therapeutic protein to a level of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of normal expression of a heterologous protein.

In certain embodiments, the methods of the invention can result in reduced expression or activity of a therapeutic nucleic acid-targeted protein.

In certain embodiments, the methods of the invention can result in a decrease in the expression or activity of a therapeutic nucleic acid-targeted protein of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the normal expression of the target protein.

Non-limiting examples of biological samples from a subject that can be analyzed include whole blood, serum, plasma, and the like, as well as combinations thereof. The biological sample may be free of cells, or may include cells (e.g., erythrocytes, platelets, and/or lymphocytes).

Any suitable method of measuring therapeutic protein or nucleic acid expression known to those of skill in the art may be used in the present invention in view of this disclosure. Exemplary methods of analyzing and measuring the expression level of a heterologous protein or nucleic acid in a biological sample include, for example, ELISA.

As used herein, the term "immune response" refers to a response of the immune system of a subject. For example, an immune response includes, but is not limited to, a detectable change (e.g., an increase) in Toll receptor activation, lymphokine (e.g., cytokine (e.g., thl or Th2 type cytokine) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., cd4+ or cd8+ T cells), NK cell activation, and/or B cell activation (e.g., antibody production and/or secretion). Other examples of immune responses include the binding of an immunogen (e.g., an antigen (e.g., an immunogenic polypeptide)) to an MHC molecule and induction of a cytotoxic T lymphocyte ("CTL") response, induction of a B cell response (e.g., antibody production), and/or a delayed-type hypersensitivity (DTH) response to an antigen from which the immunogenic polypeptide is derived, expansion of immune system cells (e.g., T cells, B cells (e.g., growth of any developmental stage (e.g., plasma cells)), and increase in processing and presentation of antigen by antigen presenting cells.

As used herein, "minimal or no undesired immune response" induced by a non-viral vector refers to a safe or therapeutically tolerable immune response in a human subject. The undesired immune response induced by the vector is different from the desired immune response of the subject induced by the antigen encoded by the non-viral vector.

In certain embodiments, the safe or therapeutically tolerable immune response is related to the level of cytokine release in the subject upon administration of a non-viral vector comprising a heterologous polynucleotide.

In certain embodiments, the methods described herein reduce the incidence of toxic cytokine release or "cytokine release syndrome" (CRS) or "severe cytokine release syndrome" (sccrs) or "cytokine storm" that may occur in a subject.

Those of skill in the art will understand that reducing toxic cytokine release or toxic cytokine levels includes reducing or inhibiting production of toxic cytokine levels in a subject, or inhibiting or reducing the incidence of cytokine release syndrome or cytokine storm in a subject. In certain embodiments, the toxic cytokine comprises a proinflammatory cytokine. In certain embodiments, the pro-inflammatory cytokine comprises IL-6, IFN-gamma, IL-1β, or TNF- α, or any combination thereof.

In certain embodiments, the cytokine release syndrome is characterized by elevated levels of several inflammatory cytokines and adverse physiological reactions in the subject such as hypotension, high fever, and cold tremor. In certain embodiments, the CRS is characterized by elevated levels of IL-6, IFN-gamma, IL-1β, or TNF- α, or any combination thereof.

In certain embodiments, the measurement of cytokine levels or concentrations as an indicator of cytokine storms may be expressed as a fold increase, a percent (%) increase, a net increase, or a rate of change in cytokine levels or concentrations. In certain embodiments, absolute cytokine levels or concentrations above a certain level or concentration may be indicative of a subject experiencing or about to experience a cytokine storm. In certain embodiments, a level or concentration of an absolute cytokine, such as that typically found in a control subject that has not undergone non-viral gene therapy, may be indicative of a method of inhibiting or reducing the incidence of a cytokine storm in a subject that has undergone non-viral gene therapy.

Those skilled in the art will appreciate that the term "cytokine level" may encompass a measure of concentration, a measure of fold change, a measure of percent (%) change, or a measure of rate of change. In addition, methods for measuring cytokines in blood, saliva, serum, urine and plasma are well known in the art.

In certain embodiments, although it is recognized that cytokine storms are associated with an elevation of several inflammatory cytokines, INF- γ levels may be used as a common measure of cytokine storms and/or a common measure of the effectiveness of cytokine storm therapies. In certain embodiments, although it is recognized that cytokine storms are associated with an elevation of several inflammatory cytokines, IL-6 levels may be used as a common measure of cytokine storms and/or a common measure of the effectiveness of cytokine storm treatment. Those skilled in the art will appreciate that other cytokines may be used as markers for cytokine storms, such as TNF- α, IB-1 α, IL-8 or IL-13.

The level of the cytokine in the subject can be analyzed, measured or determined before and/or after administration of the non-viral vector comprising the heterologous polynucleotide. The level of the cytokine in the subject may be analyzed, measured or determined before and/or after administration of the immune cell modulator. The level of cytokines in a subject may also be analyzed or measured multiple times before and/or after administration of the non-viral vector comprising the heterologous polynucleotide and before and/or after administration of the immune cell modulating agent.

Any suitable method of measuring cytokine levels known to those of skill in the art may be used in the present invention in view of this disclosure. Exemplary methods of analyzing and measuring cytokine levels in biological samples include, for example, a mesoscale delivery platform (mesoscale delivery platform, MSD).

An effective or sufficient amount may be, but need not be, provided in a single administration, may require multiple administrations, and may be, but need not be, administered alone or in combination with another composition (e.g., a pharmaceutical agent), treatment, regimen, or therapeutic regimen. For example, the amount may be scaled up as indicated by the subject's needs, the type, status and severity of the disease being treated, or the side effects of the treatment (if any). Furthermore, an effective or sufficient amount need not be effective or sufficient if administered in a single or multiple doses without a second composition (e.g., another drug or agent), treatment, regimen, or therapeutic regimen, as additional doses, amounts, or durations above and beyond such doses, or additional compositions (e.g., drugs or agents), treatments, regimens, or therapeutic regimens may be included and are thus considered effective or sufficient in a given subject. Amounts considered effective also include amounts that result in reduced use of another treatment, therapeutic regimen or regimen, such as administration of recombinant GAA for treatment of a lysosomal storage disease (e.g., pompe disease), or administration of recombinant coagulation factor protein (e.g., FVIII or FIX) for treatment of a coagulation disorder (e.g., hemophilia a (HemA) or hemophilia B (HemB)).

For pompe disease, an effective amount is an amount of GAA, e.g., that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or clearance, reduces lysosomal changes in body tissues of the subject, or improves muscle tone and/or muscle strength and/or respiratory function of the subject. For example, an effective amount can be determined by determining the kinetics of GAA uptake by myoblasts from plasma. Myoblast GAA uptake (K uptake) of about 141-147nM can be considered effective (see, e.g., mega et al, j.biol. Chem. 2012). In animal models, levels of GAA activity in plasma of greater than about 1,000nmol/hr/mL, e.g., from about 1,000 to about 2,000nmol/hr/mL, have been observed to be therapeutically effective.

For HemA and HemB, it is believed that a concentration of clotting factor greater than 1% of the concentration of factors found in normal individuals is required to convert the severe disease phenotype to a moderate disease phenotype in order to achieve therapeutic effects. The severe phenotype is characterized by joint damage and life threatening bleeding. To convert the moderate disease phenotype to a mild disease phenotype, it is believed that a concentration of clotting factor greater than 5% of normal is required.

FVIII and FIX levels in normal humans are about 150-200ng/mL plasma, but may be lower (e.g., in the range of about 100-150 ng/mL) or higher (e.g., in the range of about 200-300 ng/mL) and still be considered normal due to functional coagulation, as determined, for example, by an activated partial thromboplastin time (aPTT) one-stage coagulation assay. Thus, a therapeutic effect may be achieved such that the total amount of FVIII or FIX in the subject/person is greater than 1% of the FVIII or FIX present in the normal subject/person, e.g. 1% of 100-300 ng/mL.

The compositions may be administered to the subject as a combined composition, or separately, such as simultaneously or serially or sequentially (before or after) with the delivery or administration of a non-viral vector comprising a heterologous polynucleotide. The present invention provides combinations wherein the methods or uses of the invention are combined with any of the compounds, agents, medicaments, treatment regimens, procedures, therapies, or compositions shown herein or known to those of skill in the art. The compound, agent, drug, treatment regimen, procedure, therapy or composition may be administered or administered prior to, substantially simultaneously with, or subsequent to the administration of the non-viral vector comprising the heterologous polynucleotide to the subject.

Thus, the invention includes methods and uses that result in a reduction in the need or use of another compound, agent, drug, therapeutic regimen, treatment regimen, procedure or therapy. For example, for a coagulation disease, the therapeutic methods of the invention have therapeutic benefit if the administration of recombinant coagulation factor protein is less frequent or reduced or eliminated in a given subject to supplement endogenous coagulation factors that are absent or deficient (abnormal or mutated) in the subject. In another example, for lysosomal storage diseases, such as pompe disease, the therapeutic methods of the invention have therapeutic benefit even if the subject has been previously administered, or continues to administer less frequent or reduced doses of the recombinant viral vector comprising GAA. Thus, a reduced need or use of another treatment or therapy is included in the present invention.

An effective or sufficient amount need not be effective in the individual subjects being treated and in each subject, nor in most treated subjects in a given group or population. An effective amount or sufficient amount indicates an effectiveness or sufficiency in a particular subject, rather than a group or general population. As is typical of such methods, some subjects will exhibit a greater response, or less or no response, to a given therapeutic method or use.

The term "improvement" means a detectable or measurable improvement in a disease or symptom thereof or potential cellular response in a subject. Detectable or measurable improvements include subjective or objective reductions, decreases, suppression, repression, restriction or control of the disease or the occurrence, frequency, severity, progression or duration of complications caused by or associated with the disease, or improvements in the symptoms or underlying causes or consequences of the disease, or reversal of the disease. For pompe disease, an effective amount is, for example, an amount that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or clearance, improves muscle tone and/or muscle strength and/or respiratory function. For HemA or HemB, an effective amount is, for example, an amount that reduces the frequency or severity of acute bleeding episodes (epoode) in a subject, or an amount that reduces clotting time as measured by a clotting assay.

Accordingly, the pharmaceutical compositions of the present invention include compositions comprising an effective amount of the active ingredient to achieve the intended therapeutic purpose. Determining a therapeutically effective dose using techniques and guidance known in the art and using the teachings provided herein is well within the ability of a skilled practitioner.

The therapeutic dose depends, among other factors, on the age and general condition of the subject, the severity of the abnormal phenotype, and the strength of the control sequences that regulate the level of expression. Thus, a therapeutically effective amount in humans will be within a relatively broad range, and can be determined by a physician based on the individual patient's response to carrier-based therapies.

A composition, such as a pharmaceutical composition, can be delivered to a subject to allow for transgene expression and optionally production of the encoded protein. In certain embodiments, the pharmaceutical composition comprises sufficient genetic material to cause a subject to produce a therapeutically effective amount of the therapeutic protein or nucleic acid.

In certain embodiments, the therapeutic effect in the subject lasts for a desired period of time. Thus, in certain embodiments, the non-viral vector provides a therapeutic effect.

The composition may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The composition may be administered to a patient alone or in combination with other agents, which affects the dosage, frequency of administration, and/or efficacy.

Methods and uses of the invention include systemic, regional or local administration, or by any route, for example, by injection or infusion. Delivery of the in vivo composition may generally be accomplished by injection using a conventional syringe, although other delivery methods such as convection enhanced delivery are also contemplated (see, e.g., U.S. patent No. 5,720,720). For example, the composition may be delivered subcutaneously, epidermically, intradermally, intrathecally, intraorbitally, intramuscularly, intraperitoneally (IP), intravenously (IV), intrapleurally, intraarterially, orally, intrahepatic, transportal, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal administration. A clinician specializing in treating a patient may determine an optimal route of administration for the composition based on a number of criteria, including but not limited to: the condition of the patient and the purpose of the treatment.

The compounds, agents, medicaments, treatments, or other therapeutic regimens or regimens may be administered as a combined composition or separately, e.g., simultaneously or sequentially (before or after) with the delivery or administration of the non-viral vector. The invention thus provides combinations wherein the methods of treatment of the invention are combined with any of the compounds, agents, medicaments, treatment regimens, procedures, therapies, or compositions shown herein or known to those of skill in the art. The compound, agent, drug, treatment regimen, procedure, therapy or composition may be administered or administered prior to, substantially simultaneously with or subsequent to the administration of the non-viral vector to a patient according to the invention.

The method of the invention is suitable for losing and obtaining functions and functional genetic defects. The term "loss of function" as used herein in a genetic defect refers to any mutation in a gene, wherein the protein encoded by the gene (i.e., the mutein) exhibits a partial or complete loss of function typically associated with wild-type proteins. The term "gain of function" as used herein in a genetic defect refers to any mutation in a gene, wherein the protein encoded by the gene (i.e., mutant protein) acquires a function that is not normally associated with the protein (i.e., wild-type protein), thereby causing or resulting in a disease or disorder. The function-obtaining mutation may be a deletion, addition or substitution of one or more nucleotides in the gene, thereby causing an alteration in the function of the encoded protein. In certain embodiments, the function-gain mutation alters the function of the mutein or results in an interaction with other proteins. In certain embodiments, the function-gain mutation results in a reduction or removal of a normal wild-type protein, e.g., by an altered interaction of the mutant protein with the normal wild-type protein.

Diseases and conditions that may be treated by the methods of the invention include, for example, but are not limited to, pulmonary diseases (e.g., cystic fibrosis), hemorrhagic conditions (e.g., hemophilia a or hemophilia B with or without inhibitors), thalassemia, hematological conditions (e.g., anemia), alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic Lateral Sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartylglucosamine), barton's disease (Batten disease), advanced infant-type neuronal ceroid lipofuscinosis type 2 (late infantile neuronal ceroid lipofuscinosis type 2, cln 2), cystine storage diseases (cystinosis), fabry disease, gaucher's type I, type II and type III, glycogen storage disease II (pompe), GM 2-ganglioside disease type I (GM 2-gangliosidosis type I) (I-saxol), GM 2-ganglioside storage disease (Tay Sachs disease)), GM 2-ganglioside (hudrome), type 62, type II (sham-type I), and type III (sanatose type I, tsu-type 62) and type III (sanatose) of the late-infant type B, type III (tsugan), advanced infant type III (tsu), type III (tsu-B-tsu), type III (tsu), and type III (tsu-tsu type III), lami (maroteux-Lamy) disease and sley (sley) disease, niemann-Pick disease (Niemann-Pick disease) type a/B, type C1 and type C2, and sindler disease (Schindler disease) type I and type II), hereditary Angioedema (HAE), copper or iron accumulation disorders (e.g., wilson's disease or stokes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage disease), solid organ diseases (e.g., brain, liver, kidney, heart), or infectious viral (e.g., hepatitis B and C, HIV, etc.), bacterial or fungal diseases.

Glycogen storage disease type II, also known as pompe disease, can be treated by the methods of the invention. Pompe disease is an autosomal recessive genetic disorder caused by mutations in the gene encoding the lysosomal enzyme acid alpha-Glucosidase (GAA) which catalyzes the degradation of glycogen. The resulting enzyme deficiency results in pathological accumulation and lysosomal changes of glycogen in all tissues of the body, leading to cardiac, respiratory and skeletal muscle dysfunction (van der Ploeg & Reuser, 2008).

Coagulation disorders that can be treated by the methods of the invention include, for example, but are not limited to, hemophilia a-partner inhibitory antibodies, hemophilia B-partner inhibitory antibodies, a deficiency of any coagulation factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or FV/FVIII combination deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency or gamma-carboxylase deficiency.

Other diseases or conditions that may be treated by the methods of the invention include, for example, but are not limited to, anemia, hemorrhage associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated Intravascular Coagulopathy (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentosan (pentasacchoride), warfarin, small molecule antithrombotics (i.e., FXa inhibitors), or platelet disorders such as giant platelet syndrome (Bernard Soulier syndrome), thrombocytopenia (Glanzmann thrombasthenia), or reservoir deficiency (storage pool deficiency).

Other diseases or conditions that may be treated by the methods of the invention include, for example, but are not limited to, proliferative diseases (cancer, tumor, dysplasia, etc.), crigler-Najjar Gu Erbing and metabolic diseases such as liver metabolic diseases, friedel-crafts ataxia, infectious diseases, viral diseases (e.g., induced by hepatitis B virus or hepatitis C virus, HIV, herpes, retroviruses, etc.), hereditary diseases (cystic fibrosis, amyotrophic Lateral Sclerosis (ALS), myopathies such as Duchenne's (Duchenne) myopathy or muscular dystrophy, myotubular diseases, sickle cell anemia, sickle cell disease, fanconi's anemia, diabetes, amyotrophic Lateral Sclerosis (ALS), myotube myopathy, motor neuron diseases such as Spinal Muscular Atrophy (SMA), spinal bulbar muscular atrophy or fibular muscular atrophy (Charcot-Marie-Tooth disease), arthritis, severe combined immunodeficiency (such as RS-SCID, ADA-SCID or X-SCID), wiskott-Aldrich syndrome (Wiskott-Aldrich syndrome), X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatosis, etc.), coagulation factor deficiency, cardiovascular diseases (restenosis, ischemia, dysbiosis, homozygous familial hypercholesterol, etc.), ocular degeneration such as Leber's disease, lesion neuropathy (Lesion) and Lesion-induced neuropathy; lysosomal storage diseases such as san frani wave syndrome; hyperbilirubinemia such as CN I or II or gilbert syndrome, glycogen storage disease such as GSDI, GSDII (pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII or fatal congenital heart glycogen storage disease.

In certain embodiments, the subject has a disease affecting or originating from the Central Nervous System (CNS). In certain embodiments, the disease is a neurodegenerative disease. Non-limiting examples of CNS or neurodegenerative diseases include alzheimer's disease, huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, kennedy disease, polyglutamine repeat disease, or parkinson's disease. In certain embodiments, the disease is a psychotic disorder, addiction (e.g., tobacco, alcohol, or medication), epilepsy, kandelian disease, or adrenoleukodystrophy. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease, such as, but not limited to, spinocerebellar ataxia (SCA 1, SCA2, SCA3, SCA6, SCA7, or SCA 17).

The invention may be used in human and veterinary medical applications. Suitable subjects therefore include mammals, such as humans, as well as non-human mammals. The term "subject" refers to animals, typically mammals, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, red chimpanzees, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, adolescent and adult subjects. Subjects also include animal disease models, e.g., protein/enzyme deficient mice and other animal models, such as pompe disease (GAA loss), glycogen Storage Disease (GSD), and other diseases known to those of skill in the art.

Kit for detecting a substance in a sample

Further provided herein is a composition or combination, e.g., as a kit or kit, having (a) a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier; (b) a phagocyte depleting agent; and (c) a label having instructions for performing the methods described herein, wherein (a) and (b) are in different or the same container. Any suitable phagocyte depleting agent may be included in the combination or kit, including but not limited to a CD115 inhibitor, such as an antibody or antigen binding fragment thereof that specifically binds CD115, or a small molecule inhibitor of CD 115; ly6G inhibitors, including but not limited to anti-Ly 6G antibodies or anti-Ly 6G small molecule inhibitors; CD177 inhibitors, including but not limited to anti-CD 177 antibodies or CD177 small molecule inhibitors; CD14 inhibitors, including but not limited to anti-CD 14 antibodies or CD14 small molecule inhibitors; CD15 inhibitors, including but not limited to anti-CD 15 antibodies or CD15 small molecule inhibitors; CD11b inhibitors, including but not limited to anti-CD 11b antibodies or CD11b small molecule inhibitors; CD16 inhibitors, including but not limited to anti-CD 16 antibodies or CD16 small molecule inhibitors; CD32 inhibitors, including but not limited to anti-CD 32 antibodies or CD32 small molecule inhibitors; CD33 inhibitors, including but not limited to anti-CD 33 antibodies or CD33 small molecule inhibitors; CD44 inhibitors, including but not limited to anti-CD 44 antibodies or CD44 small molecule inhibitors; CD45 inhibitors, including but not limited to anti-CD 45 antibodies or CD45 small molecule inhibitors; CD66b inhibitors, including but not limited to anti-CD 66b antibodies or CD66b small molecule inhibitors; CD18, or inhibitors, including but not limited to anti-CD 18 antibodies or CD18 small molecule inhibitors; CD62L inhibitors, including but not limited to anti-CD 62L antibodies or CD62L small molecule inhibitors; fat emulsion; empty liposomes; bisphosphonates.

In certain embodiments, the pharmaceutical composition comprises (a) a non-viral delivery nanoparticle; and (b) a non-viral vector, wherein the non-viral vector comprises a transgene operably linked to a promoter.

Have been described in certain embodiments of the invention. However, one skilled in the art can make various changes and modifications to the invention and adapt it to various uses and conditions without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate, but not in any way limit the scope of the invention.

Examples

EXAMPLE 1 depletion of immune cells with anti-CD 115 increases transgene expression

anti-CD 115 antibodies (BioXcell, clone AFS 98) were used, which target monocytes and macrophages; anti-Ly 6G antibody (BioXcell, clone 1 A8) was used, which targets myeloid-derived cells; using a large ODN, e.g., ODN oligo, sized 200nm formulated in LNP, which inhibits TLR-9, AIM2 and other innate immunosensors; alternatively, the effect of immune depletion on cytokine release and transgene expression was studied using H-151, which is an interferon gene small molecule Stimulator (STING) inhibitor.

10 or 50 μg of transgenic DNA plasmid (nanoplasmid encoding human coagulation factor IX (hFIX) (DNA-LNP) (DNA purchased from Nature Tech) formulated in lipid nanoparticle (LNP, purchased from Precision Nanosystems) delivery vehicle was administered in mice (n=5), by Intravenous (IV) alone or Intraperitoneal (IP) administration of anti-CD 115 antibody, anti-Ly 6G antibody, or H-151 (purchased from invitogen), or 50 μg of large ODN (purchased from invitogen), as listed in table 2.

TABLE 2 design of experiments

Immune response

A set of pro-inflammatory cytokines in plasma samples 6-hours post-dosing were analyzed using MSD: IL-6, IFN-gamma, TNF-alpha and IL-1 beta. The results are shown in fig. 1A and 1B.

As shown in FIG. 1A, a reduction in induction of IFN-gamma was observed in animal plasma 6 hours after administration of 50 μg of DNA-LNP along with 50 μg of large ODN, as compared to administration of 50 μg of DNA-LNP alone.

As shown in FIG. 1B, no decrease in induction of IFN-gamma was observed in any of the animals following administration of 10. Mu.g of DNA-LNP.

Transgene expression

On days 1, 8, 14, 28, 63, 84 and 98, circulating hFIX levels in plasma samples were analyzed using ELISA.

As shown in FIG. 2A, in the absence of anti-CD 115 or anti-Ly 6G antibodies, substantially no FIX expression was detected in the plasma of animals dosed with 50 μg of DNA-LNP, whereas FIX expression of about 250ng/mL (about 5% or more of normal expression level) was observed in the plasma of animals more than 14 weeks after dosing with 50 μg of DNA-LNP in combination with anti-CD 115 antibody, and FIX expression of about 100-200ng/mL (less than 5% normal expression level) was observed in the plasma more than 14 weeks after dosing with 50 μg of DNA-LNP in combination with anti-Ly 6G antibody.

As shown in FIG. 2B, in the absence of anti-CD 115 or anti-Ly 6G antibodies, substantially no FIX expression was detected in the plasma of animals dosed with 10 μg of DNA-LNP, whereas FIX expression of about 250ng/mL (about 5% or more of normal expression levels) was observed in the plasma of animals more than 14 weeks after dosing with 10 μg of DNA-LNP in combination with anti-CD 115 antibody, and FIX expression of about 100-300ng/mL was observed in the plasma more than 14 weeks after dosing with 10 μg of DNA-LNP in combination with anti-Ly 6G antibody.

Taken together, the results demonstrate that depletion of immune cells with anti-CD 115 can increase transgene expression while reducing immune responses.

Example 2 depletion of immune cells with anti-CD 115 and another immunosuppressant increases transgene expression and decreases cytokine production

The effect of immune cell depletion on cytokine release and transgene expression was investigated using anti-CD 115 antibodies in combination with clodronate, a toxic agent encapsulated in liposomes that induced monocyte/macrophage apoptosis,

FIX transgenic DNA plasmid (50 μg) formulated in Lipid Nanoparticle (LNP) delivery vehicle (SPK-LNP 1) was administered in mice (n=5, balb/c; jackson Laboratory) by intravenous administration alone or in combination with anti-CD 115 antibodies and/or clodronate (available from Liposoma) as listed in table 3. SPK-LNP-1 has the following composition: 35% of C12-200 lipids; 16% DOPE;46.5% cholesterol; and 2.5% of C14-PEG 2000%Polar Lipids). The DNA and anti-CD 115 antibodies are as described in example 1.

TABLE 3 design of experiments

Immune response

A set of pro-inflammatory cytokines in plasma samples 6-hours post-dosing were analyzed using MSD: IL-6, IFN-gamma, TNF-alpha and IL-1 beta. As shown in fig. 3A and 3B, depletion of immune cells with anti-CD 115 antibodies or chlorophosphonate alone can moderately reduce cytokine (INF- γ and/or IL-6) levels following DNA-LNP administration, whereas combined anti-CD 115 antibodies or chlorophosphonate treatment almost eliminates cytokine induction.

Transgene expression

At week 1, circulating hFIX levels in plasma samples were analyzed using ELISA. As shown in FIG. 4, about 500ng/mL (10% of normal expression) of FIX expression was observed in animal plasma one week after administration of 50 μg of SPK-LNP1 in combination with anti-CD 115 antibody and clodronate, which was significantly higher than that of SPK-LNP1 alone or in combination with only one of anti-CD 115 antibody and clodronate.

Taken together, the results demonstrate that the dual regimen of anti-CD 115 antibody and chlorophosphonate almost eliminates the immune response while increasing transgene expression.

EXAMPLE 3 depletion of immune cells with clodronate, anti-CD 115 and pexidantinib

Balb/C mice were treated with the immune cell depleting agents chlorophosphonate, CD115 antibodies (as described in example 1) or pexidastinib. Clodronate was provided by IV administration at 50mpk (1.25 mg) or 17mpk (425 μg), and anti-CD 115 antibody was provided by IP administration at 12mpk (300 μg) 1 dose and 4 doses. anti-CD 115 antibodies are described in example 1. Pexidanib is provided by gavage (oral gavage) administration at a dose of 100mpk (2.5 mg) 4. Untreated mice groups served as controls. Each group contained 5 male BALB/c mice.

After treatment, mice were euthanized and liver samples were harvested for histology. Liver samples were stained with anti-CLEC 4F or anti-CD 68 mouse antibodies. Individual scores for the treatment groups were normalized to the untreated group to calculate depletion efficacy. The specific treatment dosage levels and schedules are shown in table 4:

TABLE 4 Table 4

The results are shown in FIGS. 5A and 5B. Clodronate and anti-CD 115 antibodies have a significant effect on kupfer (Kuffer) and macrophage depletion. Pexidanib has little effect on kupfu cell depletion (clec4f+ staining) and no effect on cd68+ macrophage depletion. The results of pexidanib are due to the mode of administration.

Drug administration of pexidasinib in combination with mouse feed provided significant depletion of clec4f+ coulofad cells and cd68+ macrophages. Pexidasatinib was mixed with mouse feed at 400mg Pex (pexidasatinib)/kg feed. Liver histology was performed 7 or 21 days after mice were fed with Pex-feed. The results are shown in fig. 6A and 6B.

EXAMPLE 4 transgenic expression of DNA-LNP-5 following treatment with pexidantinib

Pex/kg feed was mixed with the mouse feed at 400mg Pex/kg feed. In groups 2 and 3, the mice feed was replaced with Pex feed 21 days and 7 days prior to DNA-LNP-5 administration. The experimental design is shown in table 5.

TABLE 5

Mice in group 1 were fed with conventional feed and served as controls throughout the study. Mice were dosed with DNA-LNP-5 (10. Mu.g/mouse). LNP-5 (GenVoy) ^TM LNP) obtained from Precision NanoSystems inc.) and contains: 50% ionizable lipid; 10% dspc;37.5% cholesterol; and 2.5% stabilizer (PEG-lipid) (see Roces et al, pharmaceuticals, 2020 12, 1095.). The DNA was as described in example 1. The levels of hFIX in the plasma of mice were quantified using ELISA 1-4 weeks after dosing. The results are shown in FIG. 7. After DNA-LNP-5 administration, some mice in the Pex-treated group showed detectable expression. In contrast, no detectable hFIX levels were observed in mice fed with conventional feeds.

EXAMPLE 5 transgenic expression of DNA-LNP-5 following anti-CD 115 treatment

Mice were treated three times (days-5, -3 and-1) with anti-CD 115 antibody (300 μg anti-CD 115 per injection) prior to DNA-LNP-5 administration (10 μg/mouse). Mice in the control group were treated with DNA-LNP-5 alone. The DNA and anti-CD 115 antibodies are as described in example 1. The experimental design is shown in table 6.

TABLE 6

After DNA-LNP-5 administration, mice plasma levels of hFIX were quantified using ELISA. The results are shown in fig. 8, where "ns" indicates insignificant, P < 0.05 by t-test.

EXAMPLE 6 transgenic expression of DNA-LNP-3 following anti-CD 115 treatment

Mice were treated four times (days-10, -8, -6 and-2) with anti-CD 115 antibody (300 μg anti-CD 115 per injection) prior to DNA-LNP-3 administration (25 μg/mouse). Mice in the control group were treated with DNA-LNP-3 alone. DNA-LNP-3 has the following composition: 35% cKK-E12;16% dope;46.5% cholesterol; and 2.5% of PEGylated lipid C14-PEG2000. The DNA and anti-CD 115 antibodies are as described in example 1.

The experimental design is shown in table 7.

TABLE 7

After DNA-LNP-3 administration, mice plasma levels of hFIX were quantified using ELISA. The results are shown in fig. 9, wherein "ns" indicates insignificant, P < 0.05, P < 0.01 and P < 0.001 by t-test.

Transgenic expression of DNA-LNP-4 after anti-CD 115 treatment

Mice were treated four times (days-10, -8, -6 and-2) with anti-CD 115 antibody (300 μg anti-CD 115 per injection) prior to DNA-LNP-4 dosing. Mice in the control group were treated with DNA-LNP-4 alone. LNP-4 has the following composition: 50% lipid 9 (Sabnis et al Molecular Therapy 2018, vol.26, no.6, 1509-1519); 10% dspc;38.5% cholesterol; and 1.5% C-14-PEG2000. The experimental design is shown in table 8.

TABLE 8

After DNA-LNP-4 administration, mice plasma levels of hFIX were quantified using ELISA. The results are shown in fig. 10, where "ns" indicates insignificant, P <0.05 and P <0.001 by t-test.

Numerous embodiments of the invention are described herein using deterministic language to generally disclose the invention. The invention also specifically includes embodiments such as materials or materials, method steps and conditions, protocols or procedures which exclude, in whole or in part, specific objects. For example, in certain embodiments of the invention, materials and/or methods are excluded. Thus, even though the invention is not generally described herein as not being included in the invention, aspects not explicitly excluded from the invention are still disclosed herein.

Claims

1. A method of delivering a transgene to a subject in need thereof, comprising:

a. Administering a phagocytic depleting agent to the subject; and

b. administering to the subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the transgene is expressed in the subject.

3. The method of any one of claims 1-2, wherein the transgene is operably linked to a promoter or promoter/enhancer.

4. The method of any one of claims 1-3, wherein the transgene is operably linked to a liver-specific promoter or liver-specific promoter/enhancer.

5. The method of claim 4, wherein the liver-specific promoter or liver-specific promoter/enhancer is selected from the group consisting of: transthyretin (TTR) gene promoter and mutant forms thereof, human alpha 1-antitrypsin (hAAT) promoter, apolipoprotein A-I promoter, albumin promoter, hepatitis B virus core promoter, alpha Fetoprotein (AFP), human factor IX promoter, thyroxine-binding globulin (TBG) promoter, and apolipoprotein E (ApoE)/hAAT.

6. The method of any one of claims 1-5, wherein the non-viral vector is a non-viral delivery nanoparticle.

7. The method of any one of claims 1-6, wherein the non-viral vector and the phagocytic depleting agent are co-administered.

8. The method of any one of claims 1-6, wherein the phagocytic depleting agent is administered at least one day prior to the administration of the non-viral vector.

9. The method of any one of claims 1-8, wherein the phagocyte depleting agent is a CD115 inhibitor.

10. The method of claim 9, wherein the CD115 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds CD 115.

11. The method of claim 10, wherein the antibody or antigen-binding fragment thereof is ezetimibe mab, AMG820, or carbitolizumab, or an antigen-binding fragment thereof.

12. The method of any one of claims 1-11, further comprising administering a bisphosphonate to the subject.

13. The method of claim 12, wherein the bisphosphonate is clodronate, pamidronate, ibandronate, or zoledronate.

14. The method of claim 13, wherein the bisphosphonate is a chlorophosphonate.

15. The method of any one of claims 1-14, further comprising administering an immunosuppressant to the subject.

16. The method of claim 15, wherein the immunosuppressant is a steroid.

17. The method of claim 16, wherein the steroid is a corticosteroid.

18. The method of claim 17, wherein the corticosteroid is dexamethasone.

19. The method of any one of claims 1-18, wherein the undesired immune response induced by the non-viral vector by the subject is minimal or absent.

20. The method of any one of claims 6-19, wherein the non-viral delivery particle is selected from the group consisting of a lipid nanoparticle, a polymer nanoparticle, a protein-based nanoparticle, and a peptide cage.

21. The method of any one of claims 1-20, wherein the non-viral vector is a dsDNA molecule, and wherein the dsDNA molecule is selected from the group consisting of a micro-loop, a plasmid, an open linear duplex DNA, and a closed linear duplex DNA (CELiD/ceDNA/doughybone DNA).

22. The method of any one of claims 1-20, wherein the non-viral vector is a ssDNA molecule, and wherein the ssDNA molecule is selected from the group consisting of closed circular DNA and open linear DNA.

23. The method of any one of claims 1-22, wherein the transgene encodes a therapeutic or prophylactic protein or peptide.

24. The method of any one of claims 1-22, wherein the transgene encodes a therapeutic or prophylactic nucleic acid.

25. The method of any one of claims 6-24, wherein the non-viral delivery nanoparticle is a Lipid Nanoparticle (LNP) comprising one or more of the lipids provided in table 1, or a pharmaceutically acceptable salt thereof.

26. The method of claim 25, wherein the one or more lipids are present in the LNP at a mol% of about 20% to about 65%.

27. A kit, comprising:

a. a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier; and

b. phagocyte depleting agents.

28. The kit of claim 27, wherein the non-viral vector is a non-viral delivery nanoparticle and the transgene is operably linked to a promoter or promoter/enhancer.

29. The kit of claim 27 or 28, further comprising at least one of a bisphosphonate, a corticosteroid, and an immunosuppressant.

30. The kit of claim 28 or 29, wherein the phagocyte depleting agent is a CD115 inhibitor and the kit comprises:

a. a pharmaceutical composition comprising the non-viral delivery nanoparticle and the non-viral vector;

a cd115 inhibitor; and

c. at least one of clodronate, dexamethasone, and JAK inhibitor.

31. The kit of claim 30, wherein the CD115 inhibitor is ibritumomab, AMG820, or carbiruilizumab, or an antigen-binding fragment thereof.